Neuronal Nogo66 receptor-1 (NgR1) binds the myelin inhibitors NogoA, OMgp, and myelin-associated glycoprotein (MAG) and has been proposed to function as the ligand-binding component of a receptor complex that also includes Lingo-1, p75NTR, or TROY. In this study, we use Vibrio cholerae neuraminidase (VCN) and mouse genetics to probe the molecular composition of the MAG receptor complex in postnatal retinal ganglion cells (RGCs). We find that VCN treatment is not sufficient to release MAG inhibition of RGCs; however, it does attenuate MAG inhibition of cerebellar granule neurons. Furthermore, the loss of p75NTR is not sufficient to release MAG inhibition of RGCs, but p75NTR−/− dorsal root ganglion neurons show enhanced growth on MAG compared to wild-type controls. Interestingly, TROY is not a functional substitute for p75NTR in RGCs. Finally, NgR1−/− RGCs are strongly inhibited by MAG. In the presence of VCN, however, NgR1−/− RGCs exhibit enhanced neurite growth. Collectively, our experiments reveal distinct and cell type–specific mechanisms for MAG-elicited growth inhibition.
One well-characterized inhibitor of axonal growth is myelin-associated glycoprotein (MAG), an Ig superfamily member expressed by myelinating glia (Filbin, 2003). In the central nervous system (CNS), MAG is localized to periaxonal oligodendroglial membranes of myelin sheaths and participates in axon–glia interactions (Quarles, 2007). Germline ablation of MAG is not sufficient to promote axonal regeneration in vivo (Bartsch et al., 1995), but acute inactivation of MAG in retina-optic nerve cultures leads to enhanced optic nerve regeneration (Wong et al., 2003). In addition to its role as a regulator of neuronal growth, MAG has also been shown to regulate the long-term stability and integrity of axon-myelin associations in the optic nerve (Bartsch et al., 1997) and other fiber systems (Pan et al., 2005). The mechanisms that enable MAG to exert its pleiotropic effects are not well understood and are only now starting to be defined.
MAG is a member of the Siglec family of sialic acid–binding Ig-lectins with an ectodomain comprised of five Ig-like repeats (Crocker, 2002). MAG binds to the neuronal cell surface and inhibits growth in a sialic acid–dependent Vibrio cholerae neuraminidase (VCN)–sensitive manner (Kelm et al., 1994; DeBellard et al., 1996). Select gangliosides, including GD1a and GT1b, support MAG binding in a sialic acid–dependent manner, and postnatal cerebellar granule neurons (CGNs) isolated from mice lacking complex gangliosides are substantially less inhibited by MAG, indicating that gangliosides play an important role in MAG inhibitory neuronal responses (Vyas and Schnaar, 2001; Vyas et al., 2002). A soluble fusion protein of MAG comprised of the first three Ig repeats, MAG(1–3)-Fc, binds to neurons in a sialic acid–dependent manner but is not sufficient to bring about inhibition (Tang et al., 1997). This suggests that sialic acid–independent sites located in Ig repeats 4 or 5 of the MAG ectodomain are important for neurite outgrowth inhibition.
More recently, MAG has been found to interact with members of the Nogo receptor family, including neuronal Nogo66 receptor (NgR)-1 and NgR2 (Domeniconi et al., 2002; Liu et al., 2002; Venkatesh et al., 2005). NgR1 has been proposed to function as the ligand-binding component of a tripartite NgR1–p75NTR–Lingo-1 receptor complex that signals MAG inhibition (Wang et al., 2002; Yamashita et al., 2002; Mi et al., 2004). Upon MAG binding to the neuronal cell surface, p75NTR undergoes α- and γ-secretase–dependent proteolytic cleavage, and processing of p75NTR is important for RhoA activation and subsequent inhibition of neurite outgrowth (Domeniconi et al., 2005). Similar to p75NTR, the structurally related protein TROY associates with NgR1 and Lingo- 1. In the mature CNS, p75NTR expression is restricted, and TROY has been proposed to serve as a functional substitute in neurons that lack p75NTR (Park et al., 2005; Shao et al., 2005). In spite of the growing number of cell surface receptor components implicated in MAG inhibition, their role and relative contribution to growth inhibition in different cell types has not yet been examined. In this study, we provide evidence that MAG uses distinct and cell type–specific mechanisms to signal growth inhibition in different neuronal cell types, a finding that may have important implications for the development of strategies aimed at promoting neural repair after CNS injury.
Results And Discussion
Loss of terminal sialic acids attenuates MAG inhibition in a cell type–specific manner
Neurite outgrowth of postnatal retinal ganglion cells (RGCs), a population of myelinated CNS neurons, is strongly inhibited by MAG. On CHO-MAG feeder cells, Thy-1–immunopanned RGCs from postnatal day (P) 7–10 rat retina are strongly inhibited (neurite length = 10.2 ± 0.6 μm) compared with control CHO cocultures (neurite length = 26.0 ± 1.6 μm). To examine whether sialoglycans are important for MAG-mediated inhibition of RGCs, cultures were treated with increasing concentrations of VCN to remove cell surface terminal sialic acids (Fig. 1, A and B). Interestingly, neurite length on CHO-MAG cells is not significantly enhanced in the presence of VCN either at 2.5 (11.6 ± 0.7 μm; P = 0.427) or 5.0 mU/ml (11.5 ± 1.3 μm; P = 0.569) of enzyme when compared with CHO-MAG control cultures not treated with VCN (10.2 ± 0.6 μm). Increasing the VCN dose to 7.5 mU/ml inhibits the growth of RGCs and results in significantly reduced neurite length on control CHO cells (20.9 ± 1.9 μm; P = 0.012). Together, our results suggest that sialoglycan-independent mechanisms are sufficient for MAG inhibition of RGCs.
As an additional cell type to examine whether the loss of terminal sialic acids is important for MAG inhibition, we used rat P7–8 CGNs. Neurite outgrowth of CGNs cultured on CHO-MAG cells (19.0 ± 0.8 μm) is strongly inhibited compared with CGNs grown on CHO cells (38.6 ± 1.3 μm). Consistent with a previous study (Vyas et al., 2002), we found that in the presence of 7.5 mU/ml VCN, neurite length on CHO-MAG is significantly increased (25.0 ± 1.3 μm) compared with untreated CGNs on CHO-MAG (19.0 ± 0.8 μm; P < 0.001). Neurite outgrowth on CHO cells is not significantly altered in the presence (38.9 ± 1.6 μm) or absence of VCN (38.6 ± 1.3 μm; P = 0.838). Increasing the VCN dose to 15 mU/ml did not lead to any further promotion of the neurite growth of CGNs on CHO-MAG (Fig. 1, C and D; and not depicted). To assess VCN activity, we took advantage of a previous observation that NgR1 and NgR2 show a VCN-dependent drop in molecular weight (Venkatesh et al., 2005). Because of the limited yield of immunopanned RGCs, hippocampal neurons were treated for 4 h with 2.5 mU/ml VCN. Immunoblotting of lysed cells showed an ∼3-kD shift in the molecular mass of NgR1, indicating that terminal sialic acid moieties were removed successfully (Fig. 1 E). In addition, we show that VCN treatment of feeder cells does not change neuronal growth behavior (Fig. S1, A and B). Together, our results provide evidence for a neuronal cell type–specific requirement of terminal sialic acids for MAG inhibition.
Neuronal cell type–specific requirement of p75NTR for MAG inhibition
To begin to address whether previously identified protein components of the MAG receptor complex are necessary for neurite outgrowth inhibition, RGCs from wild-type and p75NTR-deficient mice were assayed for MAG responsiveness (Fig. 2). On CHO cells, the neurite length of wild-type (33.8 ± 1.8 μm) and p75NTR−/− RGCs (33.1 ± 1.7 μm) is indistinguishable. Furthermore, on CHO-MAG cells, the neurite length of wild-type (15.0 ± 0.9 μm) and p75NTR−/− RGCs (15.2 ± 1.0 μm) is very similar (Fig. 2, A and B). Statistical analysis revealed no significant difference in neurite length between the two genotypes (P = 0.929).
In a parallel approach, we examined MAG inhibition of wild-type and p75NTR−/− dorsal root ganglion (DRG) neurons. On CHO control cells, the neurite length of wild-type (79.9 ± 2.5 μm) and p75NTR−/− (78.6 ± 2.8 μm) DRG neurons is comparable. Consistent with previous studies (Wang et al., 2002; Yamashita et al., 2002), we found that postnatal DRG neurons from p75NTR mutants (64.8 ± 1.8 μm) are disinhibited on MAG compared with wild-type controls (53.4 ± 1.7 μm; Fig. 2, C and D). The disinhibition of p75NTR−/− neurons is statistically significant (P < 0.001) but still incomplete, and fiber length is decreased compared with CHO control cultures. Previously, it was reported that CGNs of p75NTR−/− and wild-type mice show strong and comparable inhibition on crude CNS myelin (Zheng et al., 2005). To expand on this study, we examined the importance of p75NTR in the MAG-mediated inhibition of CGNs. On CHO-MAG cells, wild-type (14.4 ± 0.7 μm) and p75NTR-deficient (12.0 ± 0.4 μm) CGNs are both strongly inhibited (Fig. S2, A and B). On CHO cells, the neurite length of wild-type (28.3 ± 1.0 μm) and p75NTR−/− (30.7 ± 0.8 μm) CGNs is comparable. Importantly, in the presence of the Rho kinase inhibitor Y27632 or dibutyryl-cAMP, the MAG inhibition of p75NTR-deficient CGNs is completely released (Fig. S2 B). Together, our findings suggest that MAG inhibits the growth of RGCs and CGNs through activation of the RhoA pathway in a p75NTR-independent manner.
In RGCs, TROY is not a functional substitute for
Although p75NTR is strongly expressed in postnatal CGNs, there is little, if any, p75NTR present in postnatal and adult RGCs either in steady state (Hu et al., 1998) or after optic nerve injury (Hu et al., 1999; Hirsch et al., 2000). In neurons that lack p75NTR, the TNF receptor family member TROY has been proposed to serve as a functional substitute (Park et al., 2005; Shao et al., 2005). Because TROY is expressed in RGCs (Park et al., 2005), we asked whether TROY-deficient RGCs are less responsive to MAG inhibition. As shown in Fig. 2 (E and F), neurite outgrowth of RGCs isolated from P8 TROY mutant (10.4 ± 0.8 μm) and wild-type (10.8 ± 0.8 μm) pups is strongly inhibited by MAG. No significant difference in neurite length was observed between TROY−/− (34.4 ± 2.4 μm) and wild-type (32.2 ± 1.8 μm) RGCs plated on CHO cells (P = 0.305). In a parallel experiment, we examined the MAG responsiveness of CGNs isolated from TROY−/− mice and found no significant release of growth inhibition (P = 0.689; Fig. S2, C and D). Together, these findings suggest that TROY does not serve as a functional substitute for p75NTR in RGCs and is not necessary to bring about MAG inhibition in RGCs or CGNs.
Combined loss of
NgR1 and terminal sialic acids attenuates MAG inhibition of RGCs
Next, we assessed the role of NgR1 in the MAG-mediated inhibition of RGCs (Fig. 3). When plated on CHO cells, neurite outgrowth of wild-type RGCs (29.5 ± 1.8 μm) and NgR1−/− RGCs (24.7 ± 1.4 μm) is robust, but NgR1−/− RGC neurite length is decreased by 16.3% compared with wild-type littermate controls. Although there was a statistically significant difference between wild-type and NgR1−/− RGC neurite outgrowth on control CHO cells (P = 0.006), the percent outgrowth inhibition of wild-type (57%) and NgR1−/− RGCs (60%) on CHO-MAG cells is very similar (Fig. 3 B). On CHO-MAG cells, the neurite length of NgR1−/− RGCs (9.8 ± 0.7 μm) is decreased compared with wild-type RGCs (12.0 ± 0.8 μm; Fig. 3 C), suggesting that the reduced growth of NgR1−/− RGCs is substrate independent. Thus, compared with wild-type RGCs, NgR1−/− RGCs do not display enhanced fiber growth in the presence of MAG.
To expand on this observation, we considered the possibility that MAG uses multiple and perhaps independent pathways to signal growth inhibition. Consistent with this idea, we found that the combined loss of terminal sialic acids and NgR1 in RGCs leads to a significant increase in neurite length on CHO-MAG cells compared with untreated controls (Fig. 3 C). On CHO-MAG cells, the neurite length of NgR1-null RGCs increases significantly from 9.8 ± 0.7 to 14.1 ± 1.2 μm after VCN treatment (P = 0.036). MAG inhibition of wild-type RGCs treated with VCN is not significantly attenuated (P = 0.108). Importantly, the release of MAG inhibition of NgR1-deficient neurons treated with VCN is significant but not complete. This suggests the existence of additional mechanisms for MAG inhibition in RGCs. Importantly, in the presence of the Rho kinase inhibitor Y27632 or dibutyryl-cAMP, MAG inhibition of RGCs is fully released (Fig. S3). Together, our data imply that there are multiple and independent pathways for MAG to bring about neurite outgrowth inhibition in RGCs.
Cell type–specific mechanisms for MAG inhibition
In this study, we used a mouse genetic approach combined with the enzymatic removal of terminal sialic acid moieties to dissect the molecular composition of the neuronal receptor complex that signals MAG inhibition. The principal findings of our study are as follows: (1) loss of terminal sialic acids in CGNs but not RGCs is sufficient to attenuate MAG inhibition; (2) in DRG neurons but not in RGCs or CGNs, p75NTR is important for MAG-elicited inhibition; (3) TROY-deficient RGCs and CGNs are strongly inhibited by MAG; and (4) loss of NgR1 in RGCs is not sufficient to attenuate MAG inhibition, but the combined loss of NgR1 and terminal sialic acids in RGCs attenuates MAG inhibition. Based on these observations, we conclude that MAG uses distinct and cell type–specific mechanisms to signal neurite outgrowth inhibition.
The neuronal distribution of receptor components implicated in MAG inhibitory signaling is distinct and only partially overlapping (Pioro and Cuello, 1990a,b; Lauren et al., 2003; Park et al., 2005; Venkatesh et al., 2005; Barrette et al., 2007; Satoh et al., 2007). When coupled with previous findings that MAG inhibits a broad spectrum of postnatal neurons (DeBellard et al., 1996), this implies the existence of distinct and perhaps cell type–specific mechanisms for MAG inhibition. Initially, our studies focused on RGCs, a population of heavily myelinated neurons. Remarkably, neither the loss of sialoglycans nor individual components of the NgR1 receptor complex alone is sufficient to overcome MAG inhibition in RGCs. Interestingly, when combined with VCN treatment, the loss of NgR1 in RGCs leads to a significant disinhibition on MAG substrate (P = 0.036). The release of inhibition in VCN-treated NgR1−/− RGCs is still incomplete when compared with CHO control cultures. We conclude that neither the selective loss of NgR1 nor the enzymatic cleavage of sialoglycans alone is sufficient to overcome MAG inhibition of RGCs. When combined, however, enzymatic removal of terminal sialic acids unmasks a contribution of NgR1 in MAG inhibition. This suggests that NgR1 and sialoglycans function independently and in redundant signaling pathways to bring about MAG inhibition.
Nogo receptors and terminal sialic acids participate in MAG inhibition
Select gangliosides and the neuronal glycoproteins NgR1 and NgR2 have been shown to support MAG binding in a sialic acid–dependent manner. Functional studies revealed that CGNs treated with VCN or CGNs isolated from mice deficient for complex gangliosides are more resistant to MAG inhibition (DeBellard et al., 1996; Vyas et al., 2002). Previously, it has been found that the first three Ig repeats of MAG bind to neurons in a sialic acid–dependent manner but are not sufficient to inhibit growth (Tang et al., 1997).
In spite of the broad distribution of MAG-binding gangliosides, we find that enzymatic removal of terminal sialic acids is not sufficient to attenuate the MAG inhibition of RGCs. This is in marked contrast to CGNs, a neuronal cell type in which the enzymatic removal of terminal sialic acids substantially attenuates MAG inhibition (Vyas et al., 2002). The differences in MAG inhibition observed between VCN-treated CGNs and RGCs is unexpected and suggests that MAG uses cell type–specific mechanisms to bring about inhibition. When compared with RGCs, CGNs are a population of largely unmyelinated neurons that express low levels of NgR1 and lack NgR2 protein expression (Venkatesh et al., 2005; Zheng et al., 2005). This suggests that Nogo receptors play a minor role, if any, in the MAG-mediated inhibition of CGNs. In RGCs, however, NgR1 and NgR2 are more abundantly expressed, providing a potential explanation for why the selective loss of terminal sialic acids in RGCs is not sufficient to attenuate MAG inhibition. In this model, NgR1 and NgR2 would have to participate in MAG inhibition in the absence of terminal sialic acids. Consistent with this idea, NgR1 and NgR2 both harbor multiple MAG docking sites, including sialic acid–dependent as well as sialic acid–independent sites (Venkatesh et al., 2005). We propose that in the presence of VCN, sialic acid–independent mechanisms of NgR1 and NgR2 are sufficient to bring about MAG inhibition. Furthermore, the incomplete release of MAG inhibition in VCN-treated NgR1-null RGCs may be a reflection of NgR2-dependent mechanisms. Consistent with the finding that p75NTR−/− and TROY−/− RGCs are still inhibited by MAG, NgR2 does not support the binding of p75NTR or TROY (unpublished data), suggesting novel, as yet unidentified signal transduction mechanisms for NgR2. Future studies are needed to determine whether the loss of MAG-binding gangliosides combined with the functional ablation of NgR1 and NgR2 is sufficient to fully overcome MAG inhibition in RGCs and other neuronal cell types.
Limited role of the TNF receptor family members p75NTR and TROY in MAG inhibition
The transmembrane-spanning proteins Lingo-1, p75NTR, and TROY have been identified as the signal transducing components in the NgR1 receptor complex. Upon MAG binding to the neuronal cell surface, p75NTR undergoes regulated intramembrane proteolysis, and release of the intracellular domain of p75NTR is important for RhoA activation and inhibition of neurite outgrowth (Domeniconi et al., 2005). Our functional studies with p75NTR-deficient neurons revealed that p75NTR is important in DRG neurons but not in RGCs or CGNs for MAG inhibition (Figs. 2 and S2). This suggests a more limited and cell type–specific requirement for p75NTR. Commensurate with this observation, an independent study (Zheng et al., 2005) found that p75NTR-deficient DRG neurons are more resistant to CNS myelin inhibition than wild-type littermate controls. Under similar experimental conditions, no difference in inhibition between CGNs of wild-type and p75NTR-deficient mice was observed (Zheng et al., 2005). In the present study, we expand on these findings by showing that p75NTR in DRG neurons but not in CGNs participates in MAG inhibition. It is of interest that p75NTR−/− CGNs are disinhibited on Nogo66 but not on MAG substrate (Zheng et al., 2005), This suggests that Nogo66 and MAG use distinct receptor systems to bring about growth inhibition. Because CGNs do not express the MAG-specific receptor NgR2, the mechanism used by MAG to inhibit the growth of p75NTR−/− CGNs is unknown.
RGCs express low levels of p75NTR, and, after injury to the optic nerve, p75NTR is not up-regulated in RGCs (Hu et al., 1998, 1999; Hirsch et al., 2000). Consistent with this, RGCs from p75NTR mice are strongly inhibited by MAG. TROY is expressed in RGCs and has been proposed to serve as a functional substitute in neurons that lack p75NTR (Park et al., 2005; Shao et al., 2005). However, similar to wild-type controls, TROY−/− RGCs are strongly inhibited by MAG. This suggests that in RGCs, TROY does not serve as a functional substitute for p75NTR, a cell type that normally lacks p75NTR expression. Consistent with our functional studies, it was recently reported that TROY has a restricted neuronal expression in the adult mouse CNS (Barrette et al., 2007).
Implications for axonal regeneration after CNS injury
Commensurate with its pleiotropic effects, a growing number of MAG receptors has been identified. However, their relative contribution to MAG-elicited outgrowth inhibition has not yet been examined systematically. In this study, we provide evidence for cell type–specific mechanisms for MAG-elicited growth inhibition, an observation that may have important implications for the development of strategies aimed at overcoming the growth inhibitory barrier of adult CNS myelin. We propose that depending on the CNS fiber tracts injured, cell type–specific strategies may be needed to promote axonal growth and regeneration. It has not yet been examined whether other myelin inhibitors, including Nogo-A and OMgp, use cell type–specific mechanisms to inhibit neuronal growth. The lack of robust regenerative growth in spinal cord–injured mice with germline ablation of individual receptor components (Kim et al., 2004; Song et al., 2004; Zheng et al., 2005; Cafferty and Strittmatter, 2006) or myelin inhibitory proteins (Bartsch et al., 1995; Kim et al., 2003; Simonen et al., 2003; Zheng et al., 2003) suggests that multiple and at least partially redundant mechanisms exist to bring about CNS myelin inhibition. Our studies also provide evidence that within a specific neuronal cell type, multiple and partially redundant pathways contribute to MAG inhibition. Based on these findings, the existence of one major cell surface receptor system that serves as a convergence point for multiple myelin inhibitors appears unlikely. Additional studies are needed to determine whether the combined loss of multiple receptors or the ablation of multiple growth inhibitors leads to a more robust neuronal growth response in vitro and after CNS injury in vivo.
Materials And Methods
Mice and neurite outgrowth assays
Mice mutant for NgR1 (provided by M. Tessier-Lavigne, Genentech, South San Francisco, CA; Zheng et al., 2005), p75NTR (provided by H. Federoff, Aab Institute for Biomedical Research, Rochester, NY; Lee et al., 1992), and TROY (Shao et al., 2005) have been described previously. To assay MAG-mediated growth inhibition, primary neurons were cultured on confluent monolayers of CHO cells that either express recombinant long MAG on their surface or on CHO cells lacking long MAG (Mukhopadhyay et al., 1994). P7–8 rat and mouse CGNs were purified in a discontinuous Percoll gradient (Hatten, 1985). P14 mouse DRG neurons were dissected and incubated in 0.05% trypsin and 0.1% collagenase, triturated, and cultured in SATO+ medium for 18–20 h (Venkatesh et al., 2005). RGCs were isolated by anti-Thy1 immunopanning (Barres et al., 1988). In brief, P7–10 rat or mouse retina was dissected in DME (Cellgro). Retinae were digested in 2 ml DME and 30 μl papain solution (10 U/ml; MP Biomedicals) for 30 min at 37°C. The papain was removed by three washes in DME/10% FBS. The cell suspension was then plated onto 60-mm nontissue culture-coated Petri dishes that were precoated with goat anti–mouse IgG (Jackson Immuno-Research Laboratories) and anti-Thy1.1 (Serotec, Inc.) for rat or goat anti–mouse IgM (Chemicon) and with anti-Thy1.2 (Sigma-Aldrich) for mouse RGCs. After a 1-h incubation at room temperature, unbound cells were removed by several rinses in PBS. Bound cells were lifted using 0.125% trypsin/EDTA and were washed three times in DME/10% FBS. Cells were resuspended in SATO+ medium, plated on CHO feeder layers, and cultured for 20–24 h before fixation (Venkatesh et al., 2005). Rho kinase inhibitor (Y27632; Calbiochem), dibutyryl-cAMP (Sigma-Aldrich), and VCN (Calbiochem) were used at the concentrations indicated and directly added to the culture medium at the time of plating. VCN preferentially cleaves α2-3–, α2-6–, and, to a lesser extent, α2-8–linked terminal sialic acids. The loss of sialic acids in VCN-treated neurons results in a molecular weight shift of NgR1 that was assessed by immunoblotting as described previously (Venkatesh et al., 2005).
Quantitative analysis of neurite length
Quantification of neurite outgrowth was performed as described previously (Venkatesh et al., 2005). In brief, cultures were fixed in 4% PFA and blocked in 1% horse serum and 0.1% Triton X-100 in PBS, and neurons were stained with anti–class III β-tubulin antibody (TuJ1; Promega). Images were taken using an inverted microscope (IX71; Olympus) attached to a digital camera (DP70; Olympus), and neurite length was quantified using UTHSCSA ImageTool for Windows. For quantification of neurite outgrowth, processes equal or longer to approximately one cell body diameter were measured. For all experiments, the mean and SEM of neurite length was determined from multiple independent experiments. Several independent experiments were performed for quantification of the neurite length of primary neurons isolated from the following mice: p75NTR mice (DRGs, n = 3 [+/+] and n = 3 (−/−); CGNs, n = 5 [+/+] and n = 5 [−/−]; and RGCs, n = 6 [+/+] and n = 8 [−/−]), TROY mice (RGCs, n = 3 [+/+] and n = 3 [−/−]; and CGNs, n = 2 [+/+] and n = 3 [−/−]), and NgR1 mice (RGCs, n = 5 [+/+] and n = 4 [−/−]). For quantification of the neurite length of mouse RGCs treated with 5 mU VCN, several independent experiments were performed: n = 5 (NgR1+/+) and n = 4 (NgR1−/−). RGCs from NgR1 wild-type (+/+) and heterozygous (+/−) mice behave similarly in neurite outgrowth assays and were included as controls in some experiments. All data were analyzed using one-way analysis of variance followed by Dunn's or Holm-Sidak post-hoc comparisons. All statistics were performed using SigmaStat 3.0 for Windows (Systat Software).
Online supplemental material
Fig. S1 shows that the loss of cell surface sialic acid moieties on feeder cells does not alter neuronal growth behavior. Fig. S2 shows that p75NTR and TROY are not necessary for MAG-mediated inhibition of CGNs. Fig. S3 shows that MAG inhibition of RGCs is released in the presence of Rho kinase inhibitor or dibutyryl-cAMP.
We thank D. Welch and R. Geary for excellent technical assistance, R. Schnaar (The Johns Hopkins University School of Medicine, Baltimore, MD) for CHO-MAG and CHO cell lines, H. Federoff for p75NTR mice, M. Tessier-Lavigne for NgR1 mice, and S. Mi for access to TROY mice.
This work was supported by the National Institutes of Health training grant T32 MH065181 (to O. Chivatakarn), Ruth Kirschstein Predoctoral grant NRSA F31 NS07489 (to K. Venkatesh), the New York State Spinal Cord Injury Research Program, the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation's Adelson Program in Neural Repair and Rehabilitation, the Sam Schmidt Paralysis Foundation, the Christopher Reeve Paralysis Foundation, and National Institute of Neurological Disorders and Stroke grant NS047333 (to R.J. Giger).
Abbreviations used in this paper: CGN, cerebellar granule neuron; CNS, central nervous system; DRG, dorsal root ganglion; MAG, myelin-associated glycoprotein; NgR, neuronal Nogo66 receptor; RGC, retinal ganglion cell; VCN, Vibrio cholerae neuraminidase.