LOTUS suppresses axon growth inhibition by blocking interaction between Nogo receptor-1 and all four types of its ligand

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Abstract

Axon growth inhibitors such as Nogo proteins, myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp), and B lymphocyte stimulator (BLyS) commonly bind to Nogo receptor-1 (NgR1), leading to enormous restriction of functional recovery after damage to the adult central nervous system. Recently, we found that lateral olfactory tract usher substance (LOTUS) antagonizes NgR1-mediated Nogo signaling. However, whether LOTUS exerts antagonism of NgR1 when bound by the other three ligands has not been determined. Overexpression of LOTUS together with NgR1 in COS7 cells blocked the binding of MAG, OMgp, and BLyS to NgR1. In cultured dorsal root ganglion neurons in which endogenous LOTUS is only weakly expressed, overexpression of LOTUS suppressed growth cone collapse and neurite outgrowth inhibition induced by these three NgR1 ligands. LOTUS suppressed NgR1 ligand-induced growth cone collapse in cultured olfactory bulb neurons, which endogenously express LOTUS. Growth cone collapse was induced by NgR1 ligands in lotus-deficient mice. These data suggest that LOTUS functions as a potent endogenous antagonist for NgR1 when bound by all four known NgR1 ligands, raising the possibility that LOTUS may protect neurons from NgR1-mediated axonal growth inhibition and thereby may be useful for promoting neuronal regeneration as a potent inhibitor of NgR1.

Introduction

Neurons fail to re-elongate damaged axons to their original targets in the adult central nervous system (CNS). This failure has been ascribed to repulsive axon guidance molecules (Niclou et al., 2006) and axonal growth inhibitory molecules in the glial scar (Yiu and He, 2006) and in myelin (Schwab, 2010, Yiu and He, 2006). Among these obstacles, the molecular mechanism underlying myelin-associated inhibition of axonal growth has been investigated most extensively (Schwab, 2010, Yiu and He, 2006). Nogo proteins (GrandPré et al., 2000), myelin-associated glycoprotein (MAG) (McKerracher et al., 1994), and oligodendrocyte myelin glycoprotein (OMgp) (K.C. Wang et al., 2002) have been identified as the major inhibitors of axonal growth in myelin. Nogo receptor-1 (NgR1) is the common receptor for all of these axonal growth inhibitors (Domeniconi et al., 2002, Fournier et al., 2001, Wang et al., 2002a) and is expressed in many types of neurons and their axons in the CNS (X. Wang et al., 2002). Recently, B lymphocyte stimulator (BLyS), which is a tumor necrosis factor superfamily member that is expressed in astrocytes in the CNS (Krumbholz et al., 2005), has also been identified as a functional ligand for NgR1 (Zhang et al., 2009). NgR1 forms a receptor complex with leucine-rich repeat and immunoglobulin domain-containing Nogo receptor-interacting protein 1 (LINGO-1) (Mi et al., 2004) and either the 75-kDa neurotrophin receptor (p75NTR) (Yamashita et al., 2002) or tumor necrosis factor receptor superfamily member 19 (TROY) (Park et al., 2005). These co-receptors play a role in transmitting signals to intracellular molecules such as RhoA (Mi et al., 2004, Niederöst et al., 2002, Park et al., 2005, Yamashita et al., 2002) and its effector, Rho-associated, coiled-coil containing protein kinase (ROCK) (Niederöst et al., 2002). Binding of these four glial components to NgR1 triggers signal transduction to downstream molecules via the NgR1 co-receptors to induce growth cone collapse and neurite outgrowth inhibition (Schwab, 2010, Yiu and He, 2006). This signaling pathway, which is called Nogo signaling, enormously restricts the ability of neurons to regenerate their damaged axons in the CNS (Schwab, 2010, Yiu and He, 2006).

Accumulated evidence has shown that neutralizing antibodies against Nogo (Freund et al., 2006), an NgR1 antagonist that is specific for Nogo (GrandPré et al., 2002), soluble NgR1 peptides (Li et al., 2004), and genetic deletion of Nogo (Kim et al., 2003) or NgR1 (Kim et al., 2004) promote the histological and functional regeneration of damaged CNS axons. Furthermore, triple mutation of Nogo, MAG, and OMgp exhibits greater improvement in axonal regeneration in the injured CNS compared with single mutation of Nogo (Cafferty et al., 2010). These reports suggest that inhibition of the function of multiple glial components that bind to NgR1 may more effectively improve the ability of neurons to regenerate their damaged CNS axons, although whether inhibition of BLyS function contributes to the regeneration of damaged CNS axons in vivo remains unknown.

Recently, we identified lateral olfactory tract usher substance (LOTUS)/cartilage acidic protein-1B (Crtac1B) as a novel molecule that functions in axonal bundle formation by antagonizing NgR1 function by Nogo (Sato et al., 2011). However, whether LOTUS exerts antagonistic activity on NgR1 that is bound by the other three ligands remains unknown. We show here that LOTUS suppressed axonal growth inhibition that was mediated by NgR1 function by blocking the binding of these three types of NgR1 ligands. Our findings suggest that LOTUS functions as a potent endogenous antagonist for NgR1 when bound by all the known NgR1 ligands, raising the possibility that LOTUS may overcome the failure of damaged CNS neurons to regenerate due to NgR1 function.

Section snippets

LOTUS blocks the binding of MAG, OMgp, and BLyS to NgR1

We previously showed that LOTUS overexpression together with NgR1 in COS7 cells blocks the binding of Nogo66, which is the functional domain of NogoA involved in axon growth inhibition (GrandPré et al., 2000), to NgR1 (Sato et al., 2011). To examine whether LOTUS blocks the binding of the other three NgR1 ligands to NgR1, we performed a binding assay with alkaline phosphatase (AP)-fused MAG (MAG-AP), OMgp (AP-OMgp), or BLyS (AP-BLyS) to NgR1, which were overexpressed together with LOTUS in COS7

Discussion

The function of Nogo, MAG, OMgp, and BLyS through their binding to NgR1 powerfully prevents neuronal regeneration after injury to the adult CNS (Schwab, 2010, Yiu and He, 2006). This prevention is partially attenuated by inhibition of Nogo function (Freund et al., 2006, GrandPré et al., 2002, Kim et al., 2003). Moreover, this attenuation is more effectively enhanced by inhibition of the function of multiple NgR1 ligands (Cafferty et al., 2010). Recently, we found that LOTUS blocks Nogo66

Animals

Fertilized White Leghorn eggs were purchased from Yamagishi Co. and incubated at 37 °C in a standard egg incubator. The lotus mutant mice were generated as previously described (Sato et al., 2011) and were housed in a standard mouse facility with free access to autoclaved food and water. Genotypes of the offspring of the mutant mice were assessed using polymerase chain reaction (PCR).

Throughout the experimental procedures, all efforts were made to minimize the number of animals used and their

Acknowledgments

This work was supported by a grant-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to K.T. and Y.G.) and by grants for Research and Development Project of Yokohama City University (to K.T. and Y.G.). We thank Dr. Stephen M. Strittmatter at Yale University for AP-OMgp plasmid. The authors are grateful to Drs. Fumio Nakamura, Yukio Sasaki, and Naoya Yamashita at Yokohama City University for helpful discussions and Mitsuba Matsuura for technical assistance.

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