Neuroscience Research 87 (2014) 1–7

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Review article

Structural features of the Nogo receptor signaling complexes at the neuron/myelin interface Nayanendu Saha ∗ , Momchil Kolev, Dimitar B. Nikolov Structural Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA

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Article history: Received 24 April 2014 Received in revised form 23 May 2014 Accepted 13 June 2014 Available online 20 June 2014 Keywords: Chondroitin sulfate Ganglioside LINGO-1 Nogo receptor Myelin associated glycoprotein NogoA

a b s t r a c t Upon spinal cord injury, the central nervous system axons are unable to regenerate, partially due to the repulsive action of myelin inhibitors, such as the myelin-associated glycoprotein (MAG), Nogo-A and the oligodendrocyte myelin glycoprotein (OMgp). These inhibitors bind and signal through a single receptor/co-receptor complex that comprises of NgR1/LINGO-1 and either p75 or TROY, triggering intracellular downstream signaling that impedes the re-growth of axons. Structure–function analysis of myelin inhibitors and their neuronal receptors, particularly the NgRs, have provided novel information regarding the molecular details of the inhibitor/receptor/co-receptor interactions. Structural and biochemical studies have revealed the architecture of many of these proteins and identified the molecular regions important for assembly of the inhibitory signaling complexes. It was also recently shown that gangliosides, such as GT1b, mediate receptor/co-receptor binding. In this review, we highlight these studies and summarize our current understanding of the multi-protein cell-surface complexes mediating inhibitory signaling events at the neuron/myelin interface. © 2014 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structural studies of NgR1 and NgR2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structural studies of LINGO-1 and p75 NTR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interactions of NgRs with MAG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interactions of NgRs with NogoA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interactions of NgRs with OMgp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interactions of NgRs with chondroitin sulfate proteoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interactions of NgR1 with AMIGO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interaction of NgR1 with cartilage acidic protein-1B (Crtac1B/LOTUS) and Olfactomedin (Olfm1): new players in promoting axon growth . . . . . . Interactions of NgRs with gangliosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ganglioside-mediated NgR1/LINGO-1 complex formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Spinal cord injuries are devastating because the injured central nervous system (CNS) neurons are unable to regenerate correct

∗ Corresponding author. Tel.: +1 212 639 6806; fax: +1 212 717 3135. E-mail addresses: [email protected], [email protected] (N. Saha), [email protected] (M. Kolev), [email protected] (D.B. Nikolov).

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axonal and dendritic connections largely due to a number of myelin-associated growth inhibitory factors present at the site of lesion (Filbin, 2003). The three myelin-specific inhibitors, MAG, OMgp, and Nogo-A, bind to a single receptor/co-receptor complex comprising of the Nogo receptor (NgR1), LINGO-1 and either the p75 or TROY co-receptors (Domeniconi et al., 2002; Fournier et al., 2003; Mi et al., 2004; Park et al., 2005). The signal transducer p75 is then cleaved sequentially by alpha and gamma secretase, releasing an intracellular signaling peptide (Domeniconi et al., 2005), which

http://dx.doi.org/10.1016/j.neures.2014.06.003 0168-0102/© 2014 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

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activates the Rho GTPase pathway and inhibits axonal growth. It is speculated that functionally TROY can substitute for p75 (Park et al., 2005), but it is unclear whether it can undergo similar type of proteolytic cleavage. Gangliosides are also known to play an active role in the process of neuronal inhibition upon spinal cord injury, though the precise mechanism is unclear. Interestingly, trisialoganglioside (GT1b) and disialoganglioside (GD1a) were shown to be functional binding partners of MAG and NgR1 (Collins et al., 1997). NgR1 can bind GT1b directly, which could be important for its interaction with MAG (Williams et al., 2008). Structure–function analysis of myelin inhibitors and their receptors, particularly NgRs, have provided novel information regarding the discrete steps that govern the neuronal receptor–coreceptor and the myelin inhibitor–receptor interaction. Structural studies with Nogo receptors have revealed the architecture of their leucine rich (LRR) domains (Barton et al., 2003). Biochemically, the entire ectodomains of Nogo receptors of -R1 and -R2 were shown to be necessary for interaction with MAG (Semavina et al., 2011). Additionally, it has been demonstrated that in presence of GT1b, NgR1 interacts with LINGO-1 and this preformed complex attains the ability to bind Nogo-A and p75 (Saha et al., 2011). In this review we highlight the structural and biochemical aspects of the interaction of Nogo receptors (R1 and R2) with myelin inhibitors such as MAG, Nogo A and OMgp. We also discuss the implications of recent findings, that gangliosides can mediate the binding of NgR1 to LINGO-1; in the context of spinal cord injury related inhibitory signaling.

2. Structural studies of NgR1 and NgR2 NgR1 is a glycophosphatidyl (GPI)-anchored cell-surface receptor lacking both transmembrane and intracellular domains. In addition to NgR1, two other isoforms of the receptor, NgR2 and NgR3, were identified based on sequence similarity and biochemical homology (He et al., 2003). N-terminally Nogo receptors are comprised of highly conserved eight leucine-rich repeats (LRR) flanked by cysteine rich LRRNT (leucine rich repeat N-terminal) and LRRCT (leucine rich repeat C-terminal) cap domains typical of the LRR family proteins. The LRR domains are connected to the GPI-anchor for membrane attachment via a “stalk” region (Fig. 1). Structural studies of Nogo receptor ectodomain regions [NgR1: 27–311 and NgR2: 27–310] have been reported (Barton et al., 2003; He et al., 2003; Semavina et al., 2011). The crystal structures revealed that the overall architectures of the LRR regions of NgR2 and NgR1 are very similar, resembling an elongated banana, with the region corresponding to residues 104–243 being structurally identical in the two proteins (Barton et al., 2003; Semavina et al., 2011). The LRRs have low secondary structure content, with the concave side constituting of a long parallel ␤-sheet and the convex side of loops connecting the ␤-strands. The ß-strands and loops in the LRR of NgR2 and NgR1 are very similar. However, in contrast to NgR1, NgR2 has a short ␣-helix composed of residues Thr 101Arg103 (arrow1, Fig. 2A). LRR capping regions (LRRCT and LRRNT) in NgR1 and NgR2 are stabilized by four disulphide bridges, two each at the N and C-terminus. The LRRNT of NgR2 forms a loop between two short ß-strands while the corresponding region in NgR1 lacks a defined secondary structure (arrow 2, Fig. 2A). Another significant difference between NgR1 and NgR2 lies in their glycosylation patterns (Fig. 2B). In NgR1 two asparagine residues (Asn 82 and Asn 179) are glycosylated whereas the structurally corresponding residues (Asn 83 and Asn 180) in NgR2 are not. Instead, Asn50, 93 and 236 are glycosylated in NgR2, while their structurally corresponding residues in NgR1 are nonpolar (Fig. 2B). The LRR regions of NgR1 and NgR2 carry an overall positive charge with a theoretical pI of 9. In contrast, the stalk region at

physiological pH has opposite charges in the two proteins (Barton et al., 2003; Semavina et al., 2011). The stalk sequence, comprising mainly of proline and serine/threonine, is less conserved among the NgRs. It has been suggested that that the stalk region of NgR2 is structurally different from that of NgR1 (Semavina et al., 2011). This could result in different ligand-induced structural rearrangements and different binding specificities and affinities of the different NgRs. The importance of this stalk region of NgRs has also been analyzed in the context of MAG and LINGO-1 binding (Saha et al., 2011; Semavina et al., 2011). 3. Structural studies of LINGO-1 and p75 NTR LINGO-1 is a type-1 transmembrane protein and plays a major role in oligodendrocyte differentiation and myelination. The crystal structure of the human LINGO-1 ectodomain revealed a bimodular architecture consisting of LRR and Ig-like modules. The overall LINGO-1 LRR domain structure is very similar to that of the NgR ectodomain. The structural data, as well as biophysical analysis, further show that LINGO-1 can form a stable tetramer (Mosyak et al., 2006) and this tetrameric assembly might be necessary to accommodate other components of the NgR receptor complex. The signal transducer p75NTR is a type 1 transmembrane protein. The extracellular region consists of four cysteine-rich domains (CRDs). An intracellular region spanning about 150 amino acids harbors the death domains that are implicated in apoptosis (Fig. 1) (Nykjaer et al., 2005). Two crystal structures of the p75NTR death domain revealed that they can exist either as a non-covalent asymmetric dimer or a Cys379–Cys379 disulfide linked symmetric dimer (Qu et al., 2013). These two dimer arrangements are not present in other death domain containing proteins. A structure of p75 in complex with NgR1 or LINGO-1 has not been reported yet. In its unliganded form, the p75NTR ectodomain also exists as a dimer (He and Garcia, 2004). The only complex structure of p75 available so far is that with neuotrophin-3 (NT-3), an important regulator for the survival, maintenance and differentiation of central and peripheral neurons (Bothwell, 1995). The structure shows that NT-3 forms a 2:2 symmetrical complex with p75 (Gong et al., 2008). 4. Interactions of NgRs with MAG Myelin associated glycoprotein, or MAG, was identified as a sialic acid binding lectin (Ig type) that binds to gangliosides and inhibits neuronal outgrowth (Domeniconi et al., 2002; Collins et al., 1997). The precise downstream events that lead to ganglioside mediated neuronal outgrowth inhibition are unclear. Later the Nogo receptors, NgR1 and NgR2, but not NgR3, were identified as high-affinity receptors for MAG (Venkatesh et al., 2005). The MAG extracellular region consists of five Ig-like domains (residues 17–506, Fig. 1). Deletion analysis showed that the first three Iglike domains (residues 17–325) are necessary and sufficient for binding to NgRs. The most interesting feature of the MAG–NgR interactions is that NgR1 binds MAG in a sialic acid independent manner while the NgR2–MAG binding is clearly sialic acid dependent (Robak et al., 2009). A MAG construct that comprises of the last three Ig domains (residues 234–506) does not bind NgRs or gangliosides. Interestingly this construct is sufficient to inhibit neurite outgrowth suggesting that neurons see two distinct sites on MAG, one that is responsible for the initial binding and another for inhibition (Tang et al., 1997). Cell-based and biochemical studies further demonstrate that MAG recognition by NgR1 and NgR2 is very different. The LRRCTcap domain and an adjacent 13-amino acid sequence motif in the stalk region of NgR2 are necessary and sufficient for binding

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Fig. 1. Schematic representation and domain organization of the key molecular components mediating the signaling events at the neuron–myelin interface.

to MAG. In contrast, the LRRs of NgR1 (27–310) support MAG binding with the stalk region having no effect whatsoever (Robak et al., 2009). Structurally, the LRRCT capping regions are also different in NgR1 and NgR2. A short ␣-helix comprising of residues

Gly244-Asp249 is present in NgR2 and not in NgR1. Another ␣helix, consisting of residues Asp297-Leu306 in NgR1, is split in two in NgR2 (Leu299-Thr301 and Asp304-Gln 308), thus imparting more flexibility to the adjacent stalk region (Semavina et al., 2011).

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Nogo-66 fragment are most accessible on the cell surface and available for binding to NgR1, explaining why this sub-fragment is most potent in inhibiting axonal growth. Further studies over the last few years have identified a truncated water-soluble form of Nogo66, the Nogo-54. Nogo-54 is structured and mimics Nogo-66 in terms of neurite outgrowth inhibition (Li et al., 2006, 2008). Several studies have suggested that NogoA might have different topologies with respect to the lipid bilayer, and that the hydrophobic segments flanking the Nogo-66 fragment might not be singlepass transmembrane regions. This leaves the possibility that three hydrophilic segments of NogoA might be exposed to the extracellular milieu and available for interaction with NgR1. Binding studies have indeed indicated that Nogo-A interacts with NgR1 via three segments comprising of the C-terminal 39 residues, the Nogo-66 fragment, and the N-terminal 24 residues (Lauren et al., 2007). Extensive alanine scanning mutagenesis of NgR1 identified amino acid residues that are important for binding to Nogo-A (Lauren et al., 2007). Most of these (67/68, 111/113, 133/136, 158/160, 163, 182/186 and 232/234) are located in the central part of the NgR1 LRR domain, forming a primary binding site (Lauren et al., 2007). Additionally, amino acid residues at positions 89/90, 95/97, 108, 139 and 256/259 mediate high-affinity Nogo-66 binding. Several of these positions, viz. 68, 78, 81, 89, 95, 97, 113, 120, 133, 163, 259, are not conserved in NgR2 and NgR3, explaining the highly specific Nogo-A/NgR1 interaction. Recently, an active region located in the N-terminal extracellular region of Nogo-A was found to interact with a novel binding partner, sphingosine-1-phosphate receptor 2 (S1PR2) in, in vitro and in situ co-localization experiments (Kempf et al., 2014). S1PR2 is a 7-transmembrane domain protein and binding of Nogo-A leads to activation of rhoA dependent pathway associated with inhibition of neurite outgrowth (Grünewald et al., 2009; Kempf et al., 2014). However whether NgR1 and S1PR2 mediated inhibitory pathways are interconnected or mutually independent is still unclear. Fig. 2. Structures of Nogo receptors. (A) Overlap of the structure of NgR1: 27–311 (red) and NgR2: 27–310 (green). (B) The structures of NgR1 (red) and NgR2 (green) with the Asn-linked glycosylation moieties shown as bonds (Semavina et al., 2011). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Nonetheless, ELISA-based experiments with purified components showed that compared to the isolated LRR regions, the full-length NgRs (both R1and R2) bind to the MAG ectodomain (comprising of all 5-Ig domains) with greater affinity. These results highlight the importance of the stalk regions of NgRs (Semavina et al., 2011). A co-crystal structure of MAG with NgR1 and NgR2 will delineate the precise interface of these functional complexes and the amino acid residues that play critical roles in complex formation.

6. Interactions of NgRs with OMgp Oligodendrocyte myelin glycoprotein, or OMgp, is also a GPIanchored protein with LRR domains (Wang et al., 2002, Fig. 1). Similar to NogoA, OMgp exerts its inhibitory effect by selectively binding to NgR1 but not to NgR2 or NgR3. Deletion analysis of OMgp identified the LRR domain to be mainly responsible for binding to NgR1 though the C-terminal region, comprising of serine-threonine repeats, is also capable of a weak interaction. Alanine scanning mutagenesis of NgR1 revealed that, similarly to Nogo-66, the primary binding site for OMgp resides in the concave side of the LRR domain (Lauren et al., 2007). These residues are not conserved in NgR2 and NgR3 providing basis for the specific interaction of OMgp with NgR1 and not with NgR2 or NgR3.

5. Interactions of NgRs with NogoA Nogo is a member of the reticulon (RTN) protein family. The family consists of four paralogs in mammals, including RTN-1, -2, -3, and -4. RTN4, or Nogo, consists of three splice variants namely Nogo A, B and C, which all share a conserved 66 residue extracellular domain (Grandpre and Strittmatter, 2001). This domain, known as Nogo-66, is located between two transmembrane helices that root it to the oligodendrocyte cell membrane (Fig. 1). Nogo-66 is primarily responsible for the Nogo-mediated neurite outgrowth inhibition and induces growth cone-collapse (Walmsley and Mir, 2007). CD spectral analysis and NMR data of Nogo-66 shows that it is extremely disordered in solution (Vasudevan et al., 2010). However, in the presence of lipid micelles, such as a phosphocholine surface, Nogo-66 becomes ordered (Vasudevan et al., 2010). The NMR studies also confirmed that residues 31–55 within the

7. Interactions of NgRs with chondroitin sulfate proteoglycans Chondroitin sulfate proteoglycans (CSPGs) are a different class of molecules that are upregulated in adult CNS following spinal cord injury (Rhodes and Fawcett, 2004). Chemically they are quite distinct and comprise of a protein core covalently attached to a glycosaminoglycan (GAG) side chain. GAGs are large unbranched polymers composed of repeating disaccharide units of N-acetylgalactosamine and glucuronic acid (Shen et al., 2009). Similar to the myelin inhibitors (MAG, NogoA and OMgp), CSPGmediated neuronal inhibition depends on the activation of RhoA and conventional PKCs. Initially the receptor protein tyrosine phosphatase sigma (RPTP␴) was isolated as the high affinity CSPG receptor (García Alías et al., 2009). However, the incomplete release

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Fig. 3. Schematic representation of the interactions constituting Nogo receptor signaling complexes. The interaction pattern of myelin inhibitors viz. NogoA (Nogo-66), myelin associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp) with the different Nogo receptors (R1, R2, R3) have been shown with arrowheads. Likewise, the differential interaction pattern of LOTUS and chondroitin sulfate proteoglycans (CSPG) with the Nogo receptors have been highlighted. AMIGO (AMIGO-3) interacts with NgR1 selectively and can form a functional complex with p75/TROY. An active region in Nogo-A can also interact with sphingosine-1-phosphate and trigger downstream events associated neurite outgrowth inhibition.

of CSPG-mediated inhibition in RPTP deficient neurons suggested the presence of additional receptors for CSPG. A recent comprehensive study identified the Nogo family members NgR1 and NgR3 as CSPG receptors (Dickendesher et al., 2012). Biochemical and cell based analysis further revealed that NgR2, on the other hand, does not interact with CSPGs. The LRRCT and a membrane-proximal stalk region of NgR1 and NgR3 were identified as putative binding sites for CSPGs. Knockout studies in mice and cultured neurons confirmed that both NgR1 and NgR3 bind to the GAG moiety and participate in CSPG mediated neuronal outgrowth inhibition (Dickendesher et al., 2012). 8. Interactions of NgR1 with AMIGO The expression levels of LINGO-1 do not rise in the spinal cord until 14 days post injury suggesting the possibility of other NgR1 co-receptors mediating axon growth inhibition. Consequently, AMIGO-3, a neuronal cell adhesion molecule and a member of the AMIGO family (amphoterin-induced gene and ORF), containing LRR and immunoglobin domains (Fig. 1) was identified as an alternative co-receptor for NgR1 in dorsal column lesions (Kuja-Panula et al., 2003). In vitro studies indicated that AMIGO-3 forms a functional co-receptor with NgR1 and p75/TROY in transfected cells, as well as in rat and human brain lysates, and activates RhoGTP in cells exposed to CNS myelin extracts. The results suggested that AMIGO-3 substitutes for LINGO-1 in the acute phase of the adult central nervous system injury (Ahmed et al., 2013). The crystal structure of AMIGO-1 shows that is a dimer, with the LRR regions forming the dimer interface. Sequence conservation analysis and static light scattering experiments suggest that all three AMIGO family members form dimers and the LRR-LRR interface is important for stable folding and proper functioning (Kajander et al., 2011). 9. Interaction of NgR1 with cartilage acidic protein-1B (Crtac1B/LOTUS) and Olfactomedin (Olfm1): new players in promoting axon growth LOTUS or cartilage acidic protein-1B (Crtac1B) is a 646 amino acid protein and consists of a signal peptide sequence (SP), four

phenylalanyl–glycyl and glycyl–alanyl–prolyl (FG-GAP) domains, a UnbV/ASPIC (UA) domain, an epidermal growth factor-like calcium binding (EC) domain and a transmembrane (TM) domain (Steck et al., 2007, Fig. 1). Recent studies using deletion analysis of LOTUS have conclusively shown that the two C-terminal domains consisting of UA/EC suppress Nogo-66 induced growth cone – collapse under physiological conditions, specifically in cultured chick E13 DRG (dorsal root ganglion) – neurons (Kurihara et al., 2012; Sato et al., 2011). Olfactomedin (Olfm1), a secreted glycoprotein is detected in peripheral and central nervous tissues. It consists of 454 amino acids in human and is characterized by a coiled–coiled and Olfactomedin domain at the C-terminus (Fig. 1) (Tomarev and Nakaya, 2009). Recent studies have demonstrated that Olfm1 abrogates interaction of NgR1 with its co-receptors LINGO-1 and p75 in DRG neurons thereby promoting axon growth by modulating NgR1 mediated repulsive signaling (Nakaya et al., 2012). These new findings raise the possibility that LOTUS and Olfm1 may be considered to be of therapeutic importance in the context of axon regeneration following spinal cord injury. A schematic representation of all these interactions discussed above is depicted in Fig. 3.

10. Interactions of NgRs with gangliosides Gangliosides, specifically the trisialoganglioside GT1b, are known to play a role in neuronal inhibition upon spinal cord injury. Biochemical and cell-based assays have shown that GT1b bind to MAG as well to NgR (Mehta et al., 2007). Since GT1b is equipped with two terminal sialic acids (Spiro, 1973) it is possible that these sugar moieties promote MAG binding to NgR1, thus activating the neuronal receptor/co-receptor complex en route to RhoGTPase mediated inhibition of neurite outgrowth. It was initially reported that GT1b binds primarily to the FRG motifs of MAG and NgR1 (Williams et al., 2008). However, recent studies have indicated that the stalk of NgR1 plays a significant role during ganglioside mediated NgR1/LINGO-1 complex formation (Saha et al., 2011). These novel findings and their possible implications are further discussed below.

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Nogo 66 when both molecules are present in the reaction mixture. Though the precise molecular mechanism of this interaction is still unclear, it is highly possible that once a GT1b-mediated complex is formed between NgR1, LINGO-1 and p75 on the neuronal cell-surface, the overall affinity of NgR1 for Nogo-66 is strongly enhanced (Fig. 4), presumably due to ‘clustering effect’. 12. Conclusion Structural studies of NgR1 and other components of the myelin-inhibitor/neuronal-receptor complexes have provided new insights regarding the interactions that govern spinal cord injury and paralysis. The precise molecular details of these important signaling pathways will be delineated further by future studies of the three-dimensional structures of NgRs in complex with their coreceptors and inhibitory ligands. An important goal is to develop small molecule inhibitors and monoclonal antibodies that successfully disrupt or significantly modulate the formation of these complexes and promote neuronal regeneration following spinal cord injury and paralysis. Similar approaches are already underway in the context of traumatic brain injury (TBI), where pharmacological modulation of p75 by LIM11A-31, a blood brain barrier permanent small molecule enhanced the production of mature neurons from adult hippocampal neural progenitors (Shi et al., 2013). Acknowledgement The research was supported by a New York State Spinal Cord Injury Research Program grant (C-022047) to N.S. The authors declare no competing interest. References

Fig. 4. Ganglioside mediated association (clustering) of myelin inhibitors (NogoA) with neuronal receptor/co-receptor complexes (NgR1/LINGO-1/p75).

11. Ganglioside-mediated NgR1/LINGO-1 complex formation Solution based studies with purified myelin inhibitor–receptor components unfolded a novel role for gangliosides in mediating NgR1/LINGO-1 complex formation. Specifically, it was revealed that in the presence of the trisialoganglioside GT1b, NgR1 but not NgR2 forms a complex with LINGO-1. The results highlight the importance of the terminal sialic acids of the ganglioside and the stalk regions of NgR1 as the principal determinants of this receptor–co receptor interaction (Saha et al., 2011). The ganglioside GT1b also showed evidence of weak binding to the FRG motifs of NgR1. However for efficient NgR1/LINGO-1 complex formation, the entire ectodomains of the proteins were required. Notably, when the oligosaccharide chain is extended, the twocarboxyl groups in the sialic residues of GT1b are located about 15–20 A˚ from the membrane surface (He and Garcia, 2004). The terminal sialic acid, therefore, is ideally positioned interact in cis with the ectodomain of NgR1 and modulate its interaction with LINGO-1. This type of cis interaction between a protein and GT1b has previously been reported for the leukocyte cell surface antigen CD38 (Hara-Yokoyama et al., 2001). However, the NgR1/LINGO-1/GT1b complex is the first documented case of a carbohydrate moiety mediating the interaction between a cell-surface receptor and its co-receptor. The tripartite receptor/co-receptor complex comprising of NgR1, LINGO-1 and GT1b interacts strongly with p75 and

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myelin interface.

Upon spinal cord injury, the central nervous system axons are unable to regenerate, partially due to the repulsive action of myelin inhibitors, such a...
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