INVESTIGATIONS ON THE TISSUE DISTRIBUTION, LOCALIZATION AND FUNCTIONS OF BRAIN-ENRICHED LEUCINE-RICH REPEATS (LRR) CONTAINING PROTEINS AMIGO AND NGR2 CHEN YANAN NATIONAL UNIVERSITY OF SINGAPORE 2007 INVESTIGATIONS ON THE TISSUE DISTRIBUTION, LOCALIZATION AND FUNCTIONS OF BRAIN-ENRICHED LEUCINE-RICH REPEATS (LRR) CONTAINING PROTEINS AMIGO AND NGR2 CHEN YANAN B.Sc.(Hons), NUS A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE II Acknowledgements I would like to extend my grateful appreciation to all that have helped me in their unique ways throughout master program I thank my supervisor, Dr Tang Bor Luen for his scrupulous and brilliant supervision, for guiding me step by step, training me for more than four years and for his patience to review my thesis draft numerous times I would also like to thank him to be a good model of a dedicating and critical scientist I am grateful to my lab members, Ee Ling, Felicia, Catherine, Wan Jie, Qin Fen and ex-colleagues Wang Ya, Choon Bing for friendship Thank them for their cooperation, helpful discussions and technical troubleshoots It is joyful to work with them Thanks to my parents who are always there and support me Lastly, I would like to express my deepest appreciation to my husband, Yong Hong, who changes me and transforms me Without him, mission is impossible III Table of contents Abstract VIII List of Figures X List of Tables .XII Abbreviations XIII Introduction 1.1 LRR domain containing proteins 1.2 Amphoterin-induced gene and ORF (AMIGO) .8 1.3 The Nogo-66 receptor family and Nogo receptor homologue (NgR2) 10 1.4 1.3.1 Inhibition of axonal regeneration and glia scar formation in CNS injury 10 1.3.2 Overview of Nogo-66 receptor mechanism for myelin inhibition .11 1.3.3 Nogo-66 receptor and family members 14 1.3.4 The myelin-associated inhibitor (MAI) Ligands: MAG, Nogo and OMgp 17 1.3.5 The coreceptors of NgR1: p75NTR and TROY/TAJ .18 1.3.6 The NgR1 coreceptor LINGO-1 and its functions .19 1.3.7 The intracellular signaling pathway from NgR1 22 Rationale for current work 24 Materials and Methods 26 2.1 General Materials and reagents 27 2.1.1 General materials and reagents 27 IV 2.2 Plasmids construction 28 2.2.1 2.3 Expression constructs 28 Mammalian cell culture .31 2.3.1 Cell culture 31 2.3.2 Transfection and selection of stable clones 31 2.3.3 Primary cortical neuron culture .33 2.3.4 Primary glia culture .34 2.3.5 Assessment of Neurite outgrowth 34 2.3.6 AMIGO silencing in cortical nuerons 35 2.3.7 Western blot, immunofluorescence and immunohistochemistry 36 2.3.8 Antibody blocking 38 2.3.9 Confocal microscopy .38 2.3.10 Immunoprecipitation 38 2.3.11 PI-PLC treatment 39 2.4 2.5 Generation of Recombinant DNA and proteins 39 2.4.1 Strains and Growth comditions 39 2.4.2 Recombinant DNA methods 39 2.4.3 Recombinant protein preparation and analysis 41 Rabbit polyclonal antibody preparation 42 Results: AMIGO is expressed in multiple brain cell types and may regulate dendritic growth 44 V 3.1 AMIGO is expressed in multiple brain cell types 45 3.1.1 Expression of AMIGO in rodent brain 45 3.1.2 Expression of AMIGO in primary cultured neurons and glia cells .47 3.2 Polarized neuronal surface localization of AMIGO 55 3.3 The role of AMIGO in dendritic outgrowth 59 Results: NgR2 expression in the brain and investigations on its co-receptor interaction 61 4.1 4.2 Expression analysis of NgR2 .62 4.1.1 NgR2 expression in mammalian cells 62 4.1.2 Comparative analysis of NgR1 and NgR2 in mouse central nervous system65 Expression and localization of NgR family members and LINGO-1 in primary cortical neurons 72 4.3 NgR2 interacting with NgR1 co-receptors LINGO-1 and p75NTR in Neuro2A cells 74 Discussion 82 5.1 AMIGO expression and possible functions in the adult CNS 83 5.1.1 AMIGO is expressed in multiple brain cell types 83 5.1.2 Neuronal subcellular localization of AMIGO 85 5.1.3 The role/effect of AMIGO in neurite growth 87 5.1.4 Other possible roles of AMIGO in neurons 88 VI 5.2 NgR2 expression in the adult CNS and its possible role in neuronal regeneration 88 5.3 5.2.1 The MAI- NgR1 axis in inhibition of neuronal regeneration 88 5.2.2 NgR2 expression pattern in the adult mouse brain in comparison with NgR191 5.2.3 NgR2’s role and mechanism in neurite growth inhibition 93 Concluding remarks 95 References 97 Appendices .109 VII Abstract Leucine-rich repeats (LRR) are protein-protein interaction domains of 20-29 amino acid residues in length, found in proteins with diverse structure and functions An emerging group of surface proteins with an ectodomain containing LRR repeats and motifs were found to be interestingly and specifically enriched in the central nervous system (CNS) AMIGO (Amphoterin-induced gene and ORF) is a type one transmembrane protein with an ectodomain containing six LRRs, followed by a single immunoglobulin (Ig)-like domain located adjacent to the transmembrane domain AMIGO has two paralogues, named AMIGO-2/Alivin and AMIGO-3 Immunoblot, immunohitochemical and immunofluorescence analysis with antibodies raised against the short cytoplasmic region of AMIGO showed that AMIGO protein levels are developmentally regulated and is present in multiple cell types in the brain Primarily enriched in multiple neuronal subtypes, distinct staining signals could also be found in astrocytes and oligodendrocytes (both in tissue sections and in culture) Neuronal AMIGO is targeted to both axons and dendrites The subdomains of AMIGO’s ectodomain, however influences its polarized targeting in primary cortical neurons Exogenously expressed full length (AMIGO-FL) and Ig domain-deleted AMIGO (AMIGO-LRR) localize to preferably to dendrites, while a LRR-deleted (AMIGO-Ig) mutant is preferentially targeted to axons When expressed in Neuro2A neuroblastoma cells, cell surface expression of AMIGO-Ig is immediately prominent Both AMIGO-FL and AMIGO-LRR however assume a more intracellular VIII morphology Silencing of AMIGO expression for appeared to retard dendritic growth of primary cortical neurons The Nogo-66 receptor (NgR2) is a LRR containing, glycosylphosphatidyl inositol (GPI)-anchored surface protein which is a paralogue of the better known and studied Nogo-66 receptor (NgR1), which is the receptor of myelin-associated axonal growth inhibitor in adult CNS myelin NgR1 is known to function through its association with LINGO-1, which like AMIGO, has multiple LRR repeats and a single Ig-like domain Co-immunoprecipitation (Co-IP) experiments suggest that NgR2 also interacts with LINGO-1 as well as p75NTR, another known NgR1 co-receptor After induction of neurite growth with retinoic acid, neurite extension and cell adhesion of Neuro2A cells co-expressing both NgR2 and LINGO-1 grown on MAG-Fc coated coverslips were greatly impaired However, co-expression of NgR2 and a dominant-negative form of LINGO-1 have no such neurite growth inhibitory effect Our results showed that NgR2 could transducer a neurite growth inhibitory signal by engaging LINGO-1 Collectively, work reported in this thesis sheds new light on two brain-enriched LRR domains containing proteins that have, generally, opposite effects on neurite growth Studies along these lines would be expected to provide basic information that is clinically useful against neurological diseases IX List of Figures Figure 1-1 Nearest neighbour dendrogram (generated by the MegAlign program of DNASTAR) of the CNS-enriched, LRR domain -containing proteins Figure 1-2 Schematic diagram showing the domain organization of representatives of the group of LRR-containing proteins with cell-adhesion molecule-like domains Figure 1-3 The axon regeneration inhibition pathway through NgR1/NgR2 complex 13 Figure 1-4 Multiple sequence alignment (ClustalW) and schematic structural illustration of of human NgR1, NgR2 and NgR3 15 Figure 2-1 A schematic diagram showing the construct myc-NgR2 in pCIneo based on the modified vector pCIneo-SS-myc 29 Figure 3-1 Characterization of an antibody raised against AMIGO and developmental expression survey of AMIGO in mouse brain 46 Figure 3-2 Expression of AMIGO in adult mouse neurons 50 Figure 3-3 AMIGO expression pattern at the hippocampus 50 Figure 3-4 AMIGO expression in astrocytes and oligodendrocytes 53 Figure 3-5 AMIGO expression examined in primary cultures of neurons and glia 53 Figure 3-6 AMIGO expression in cultured primary cortical neurons in vitro 54 Figure 3-7 Differential expression patterns of AMIGO and its truncation mutants in Neuro2A cells 56 Figure 3-8 AMIGO and its truncation mutants label neurite with different lengths when expressed in primary cortical neurons 57 Figure 3-9 AMIGO-FL and AMIGO-LRR are localized to MAP-2-positive dendrite, but not AMIGO-Ig 59 Figure 3-10 siRNA silencing of AMIGO attenuates dendrite outgrowth of primary cortical neurons 61 Figure 4-1 NgR2 antibody specificity and NgR2 subcellular localization in Neuro2A cells 66 X B RA I N RE SE A R CH RE V I EW S 51 ( 20 ) 5–2 2002), which are largely secretory, and the more classical transmembrane Ig-superfamily of cell adhesion molecules, or Ig-CAMs (Walsh and Doherty, 1997) that are brainenriched but without LRR repeats in their ectodomains Another possible member of the group is the brainenriched LRIG-1 (or LIG-1) (Suzuki et al., 1996) and its ubiquitously expressed paralogues (Guo et al., 2004) LRIG-1 (and its likely Drosophila homologue Kekkon; Musacchio and Perrimon, 1996) binds to the epidermal growth factor (EGF) receptor and inhibits its signaling (Gur et al., 2004; Laederich et al., 2004) However, LRIG-1's deletion resulted in no apparent neuronal defects Instead, LRIG-1 deficient mice develop a skin condition after birth that is phenotypically reminiscent of the skin disease psoriasis (Suzuki et al., 2002) The proteins discussed above also did not include those transmembrane proteins found in the brain that has LRR repeats, but without any Ig-like or FN-III domains Prominent members of the latter group include the brain-enriched LGI-1 (a gene implicated in epilepsy and tumor suppression (Chernova et al., 1998; Gu et al., 2005) and its more ubiquitously expressed homologues (Gu et al., 2002)) Lib is identified as a gene upregulated by beta-amyloid treatment of rat astrocytes (Satoh et al., 2002), but is actually most enriched in placenta Another family of brain-enriched LRR repeat containing transmembrane proteins is the LRRTMs (Lauren et al., 2003b) LRRTMs (except for LRRTM4) are peculiar in the genomic sense that they are located in the introns of different alpha-catenin genes, and that these two gene families may have coevolved An emerging group of brain-enriched, LRR repeat and cell adhesion motif containing transmembrane proteins—interactions and signaling Collectively, the above described appear to be a group of proteins with similar functional domains but are heterogeneous in their physiological roles in the CNS By virtue of the presence of extracellular Ig-like and FN-III domains, the group of proteins discussed above might function in modulating neuronal cell adhesion Such cell–cell or cell substratum adhesions are important for both dynamic developmental processes such as axonal pathfinding, as well as architectural purposes such as organizational stability of the synapses or axoglial junctions The Ig-like domain in particular is the signature motif for the immunoglobulin superfamily of adhesion molecules (Kamiguchi and Lemmon, 2000) The presence of Ig-like domains, in tandem with the LRR domains, potentially broadens the repertoire or enhances the potential for a wide spectrum of multiple protein–protein interactions by members of this family However, only members of the AMIGO/Alivin have been shown to interact homotypically and heterotypically with each other in trans (i.e between two cells) The interaction between NGL-1 and netrin G should also be in trans, but this is more likely to serve a guidance rather then an architectural, cell-adhesion role LINGO-1's interaction with NgR and p75NTR or TAJ/TROY is likely in cis (i.e on the same plasma membrane), as would FLRT3's interaction with FGFR For 271 LINGO-1, the formation of the ternary LINGO-1/NgR/p75NTR complex on neuronal growth cones is essential for inhibition of neurite growth, but whether the same complex is necessary for modulation of oligodendrocyte differentiation and myelination has not yet been demonstrated It is yet unclear if any of these proteins have high affinities for extracellular matrix components, and if any of them have a role in cell–matrix interaction and inside-out or outside in signaling NLRRs and FLRTs with FN-III domains could potentially interact with integrins, but this has not been clearly demonstrated It is interesting to note that within the same molecule, the LRR motifs could potentially modulate the functional interaction of the Ig-like domains with other proteins and vice versa We have, for example, observed that these motifs, when overexpressed individually, have noticeable differences in their effects on cell morphology (Aulia and Tang, unpublished results) Future explorations would undoubtedly reveal more on the interactive partnerships of these proteins with others The particularly interesting property of these proteins, which functionally cluster them into a noticeable group, is their potential role in modulating neurite growth In this respect, there is much room for further investigations, as only the downstream signaling of LINGO-1 and FLRT3 is known to any extent Clearly, all these proteins could either signal intracellularly through their cytoplasmic domains or by engagement of other transmembrane proteins in a signaling complex The cytoplasmic domains of the AMIGO/alivin paralogues and NGL-1 paralogues are rather divergent from one another and motif scans reveal no known signaling motif The LINGO paralogues have a putative tyrosine kinase phosphorylation site that is conserved in LINGOs1–3, but not LINGO-4 It is yet unclear if this tyrosine residue is indeed phosphorylated in a regulated manner in vivo NLRR-1 and NLRR-3 have a conserved endocytosis motif at the cytoplasmic tail that is not present in NLRR-2 The latter has instead a putative WW domain, a protein interaction motif binding to polypeptide stretches that are proline-rich (Ilsley et al., 2002) The FLRTs have a conserved tyrosine kinase phosphorylation site at their cytoplasmic domain which could be a potential substrate for FGFR Of the protein families emphasized above, only LINGO-1 and NLRR-4 have been analyzed at the level of targeted gene disruption It turns out that although LINGO-1 is not essential for embryonic development, it undoubtedly has some roles to play in postnatal development of the nervous system (as gleaned from the fact that LINGO-1 deficient oligodendrocytes mature and differentiate faster) (Mi et al., 2005) NLRR-4 has a subtle, but phenotypic memory impairment that is highly interesting and which warrants further investigations A combination of gene knockout and nervous system-specific transgenic overexpression of these molecules, alone and in combinations, would yield much more information on their interactions and functions Epilogue Elaboration of the vertebrate CNS had likely benefited from the expansion and evolution of these complex arrays of signaling molecules, forming an intricate web of growth and 272 B RA I N R E SE A R CH RE V I EW S 51 ( 20 ) 5–2 differentiation control A systematic and integrated study of the functions and mechanism of function of this emerging group of transmembrane proteins with LRR repeats and cell adhesion molecule motifs would clearly provide useful insights to CNS physiology and pathology If the recent elucidation of involvement of leucine-rich repeat kinase (LRRK2)/dardarin in Parkinson disease (Singleton, 2005) is anything to go by, we could expect to hear much more about LRR repeat-containing 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leucinerich repeat protein that increases the intensity of pleiotropic cytokine responses Biochem Biophys Res Commun 305, 981–988 Wong, K., Park, H.T., Wu, J.Y., Rao, Y., 2002a Slit proteins: molecular guidance cues for cells ranging from neurons to leukocytes Curr Opin Genet Dev 12, 583–591 Wong, S.T., Henley, J.R., Kanning, K.C., Huang, K.H., Bothwell, M., Poo, M.M., 2002b A p75(NTR) and Nogo receptor complex mediates repulsive signaling by myelin-associated glycoprotein Nat Neurosci 5, 1302–1308 Yin, Y., Miner, J.H., Sanes, J.R., 2002 Laminets: laminin- and netrin-related genes expressed in distinct neuronal subsets Mol Cell Neurosci 19, 344–358 Yiu, G., He, Z., 2003 Signaling mechanisms of the myelin inhibitors of axon regeneration Curr Opin Neurobiol 13, 545–551 Zeitz, C., Scherthan, H., Freier, S., Feil, S., Suckow, V., Schweiger, S., Berger, W., 2003 NYX (nyctalopin on chromosome X), the gene mutated in congenital stationary night blindness, encodes a cell surface protein Invest Ophthalmol Visual Sci 44, 4184–4191 Zhang, Q., Wang, J., Fan, S., Wang, L., Cao, L., Tang, K., Peng, C., Li, Z., Li, W., Gan, K., Liu, Z., Li, X., Shen, S., Li, G., 2005 Expression and functional characterization of LRRC4, a novel brain-specific member of the LRR superfamily FEBS Lett 579, 3674–3682 Molecular Neurobiology Copyright © 2006 Humana Press Inc All rights of any nature whatsoever reserved ISSN 0893–7648/06/34(2): 000–000/$30.00 ISSN (Online) 1559–1182 Myelin-Associated Glycoprotein-Mediated Signaling in Central Nervous System Pathophysiology Yanan Chen, Selina Aulia, and Bor Luen Tang* Department of Biochemistry, Yong Loo Lin School of Medicine National University of Singapore, Singapore Abstract The myelin-associated glycoprotein (MAG) is a type I membrane-spanning protein expressed exclusively in oligodendrocytes and Schwann cells It has two generally known pathophysiological roles in the central nervous system (CNS): maintenance of myelin integrity and inhibition of CNS axonal regeneration The subtle CNS phenotype resulting from genetic ablation of MAG expression has made mechanistic analysis of its functional role in these difficult However, the past few years have brought some major revelations, particularly in terms of mechanisms of MAG signaling through the Nogo-66 receptor (NgR) complex Although apparently converging through NgR, a readily noticeable fact is that the neuronal growth inhibitory effect of MAG differs from that of Nogo-66 This may result from the influence of coreceptors in the form of gangliosides or from MAG-specific neuronal receptors such as NgR2 MAG has several other neuronal binding partners, and some of these may modulate its interaction with the NgR complex or downstream signaling This article discusses new findings in MAG forward and reverse signaling and its role in CNS pathophysiology Index Entries: Axonal regeneration; myelin; myelin-associated glycoprotein; Nogo-66 receptor (NgR); p75NTR vous system (CNS) and peripheral nervous system (PNS) myelin sheaths Alternatively, splicing results in two isoforms: small (S-; 582 residues) and large (L-; 626 residues) MAG (1) In rodents, L-MAG levels peak during CNS development and decline in adulthood, but SMAG is the predominant form in both adult PNS and CNS (2) Conversely to rodents, the human L-MAG splice variant predominates in adult human brain, whereas human S-MAG is Introduction The myelin-associated glycoprotein (MAG) is a type transmembrane protein found in the peri-axonal membrane of both the central ner- Received August 19, 2005; Accepted June 14, 2006 *Author to whom correspondence and reprint requests should be addressed.: bchtbl@nus.edu.sg Molecular Neurobiology Volume 34, 2006 most abundant in the PNS (3) Both forms differ only in the cytoplasmic C-terminus but have the same N-terminal extracellular domain, which contains five immunoglobulinlike motifs MAG is a member of the sialic acid-dependent immunoglobulin-like family member lectins (siglec) family (4) Because MAG is designated siglec-4a, much, but not all, of its interaction with neurons occurs in a sialic-acid-dependent manner (5) Despite being initially described as a protein that confers neural adhesion and neurite outgrowth function (6), it soon became apparent that MAG is a myelin-associated inhibitor of neurite outgrowth and axonal regeneration (5,7–9), at least in vitro and when examined using adult neurons Recombinant MAG coated onto beads induces hippocampal neuron growth cone collapse (9) Proteolytically generated, soluble MAG consisting of the extracellular domain is also a potent inhibitor of neurite outgrowth (10,11) However, analysis using MAG knockout mice (12) has not provided strong support to the notion of MAG as a major CNS neurite growth inhibitor There was no apparently significant difference in cell spreading, neurite elongation, or growth cone collapse of several cell types when myelin preparations from either MAG-deficient or wild-type mice were used as a substrate Furthermore, the extent of axonal regrowth in optic nerve and corticospinal tract lesions in vivo was equally poor in MAG-deficient and wild-type mice Because axonal regrowth in MAG-deficient mice could be similarly enhanced in wild-type mice by the Nogo-targeting antibody IN-1, it appears that MAG does not carry the majority of the neuronal growth inhibitory activity of myelin Earlier investigations using oligodendrocyte neuron cocultures and the analysis of MAGdeficient mice also suggested a role for MAG in the initiation of CNS myelination as well as the long-term preservation of myelin sheaths (extensively reviewed in ref 13) Again, however, the subtlety of the phenotypes observed made it difficult to view MAG as a major functional component of myelin It is obvious that Molecular Neurobiology Chen et al other oligodendroglia components could functionally cover for the complete absence of MAG during development MAG maybe involved in reverse signaling processes in oligodendrocytes, because crosslinking of MAG with antibodies activates Fyn tyrosine kinase (14,15) The latter phosphorylates the cytoplasmic domain of L-MAG, which is the longer splice isoform specifically involved in CNS myelination (16), principally at tyr 620 (17) Mild hypomyelination was observed in optic nerves of MAG-deficient mice This is worse in Fyn-deficient mutants, and the severity of the defects were additive in MAG/Fyn double mutants (18), suggesting that although both MAG and Fyn are important for the initiation of myelination, they may nonetheless act independently The past yr had provided substantial breakthroughs in our understanding of the forward signaling of MAG In 2002, MAG was shown to interact with the leucine-rich repeat (LRR)-containing, GPI-linked Nogo-66 receptor (NgR) (19), which transduces its growth inhibitory effect on neurons (20,21) The latter was initially cloned as a neuronal receptor mediating the neurite outgrowth inhibition by the extracellular 66-amino acid loop (termed Nogo-66) common to all major splice isoforms of Nogo (22), the target of the neurite growthenhancing IN-1 antibody (23) Another myelinassociated protein, oligodendrocyte-myelin glycoprotein (OMgp), also inhibits neurite growth through the NgR The NgR transduces a growth inhibition signal by engaging the transmembrane p75NTR (24,25) or TAJ/TROY (26,27) in conjunction with another LRR-containing transmembrane protein known as LINGO-1 (28) Signaling through p75NTR ultimately leads to the activation of Rho and its effectors, resulting in changes of the actin cytoskeleton that underlie growth cone collapse or repulsion (29) In the midst of these new developments, it should be emphasized that the effect of MAG on neurite growth is qualitatively different from that of Nogo-66 This article reviews new findings on MAG’s forward inhibitory signalVolume 34, 2006 MAG-Mediated Signaling in the CNS ing to neurons, highlighting the differences with that of Nogo-66 and citing references to MAG’s neuronal binding partners It then briefly surveys some new implications of MAG in CNS pathology AU: Consider making this and the next heading subheads? MAG’s Neuronal Interacting Partners I: Sialoglycolipids In the CNS, MAG is localized to myelin membranes juxtaposed to axons (30), and purified MAG protein incorporated into liposomes binds neuronal processes (6) Although much of the recent excitement has focused on MAG’s interaction and action through the NgR, the latter is not the only neuronal molecule interacting with MAG A prominent feature of MAG–neuron interaction is dependency on sialic acid, a feature that is not apparent in earlier reports of MAG–NgR interaction (20,21) MAG binds best to 2,3-linked sialic acid on a Gal(β1→3)GalNac core structure (31,32) Interestingly, MAG’s sialic acid binding site is actually distinct from its neurite inhibitory activity This was elegantly illustrated as a truncated form of soluble recombinant MAG (MAG-Fc; containing Ig domains 1, 2, and but missing domains and 5, fused to the immunoglobulin heavy-chain), which was bound to neurons in a sialic-acid-dependent manner but did not inhibit neurite outgrowth like full-length MAG Mutation of arginine 118 (R118) in MAG to either alanine or aspartate abolished its sialic-acid-dependent binding However, R118-mutated MAG retained a weakened capacity of inhibiting neurite outgrowth and remained a potent inhibitor when expressed at cell surfaces rather than being added as a soluble protein (33) The sialic acid moiety alluded to earlier is often found on a class of glycosphingolipids known as gangliosides, and MAG has been shown to bind a limited set of structurally related gangliosides known to be expressed in myelinated neurons These include the major brain ganglioside GD1a and GT1b, as well as a minor ganglioside GQ1bα, expressed on cholinergic neurons (34–36) Gangliosides Molecular Neurobiology mediate MAG’s inhibition of neurite outgrowth from primary rat cerebellar granule neurons, because the latter is clearly attenuated by neuraminidase treatment of the neurons, blocking of neuronal ganglioside biosynthesis, or antiganglioside monoclonal antibodies Furthermore, multivalent clustering of GD1a or GT1b using precomplexed antiganglioside antibodies mimicked MAG’s inhibitory effect (35) An earlier study showed that antibody-crosslinking of cell surface GT1b, but not GD1a, mimicked the effect of MAG Notably, the Rho kinase (ROCK) inhibitor Y27632 blocked inhibition of neurite outgrowth by both MAG and anti-GT1b antibody Activation of Rho-ROCK is a universal convergent point downstream of neurite outgrowth inhibitors, including all the major neuronal guidance ligand-receptor systems (37) Therefore, the gangliosides appeared to be an authentic neuronal MAG receptor mediating its neurite growth inhibitory function Because gangliosides are not membranespanning structures with a sizable cytoplasmic domain, they are incapable of relaying a signal to the cytoplasm The initial picture of MAG signaling through gangliosides is that MAG binding causes ganglioside clustering in lipid rafts, which somehow results in the activation of signaling proteins found in such membrane microdomains In other words, MAG-induced ganglioside clustering presumably leads to the activation of a coreceptor(s) It is apparent that p75NTR is a promising candidate for such a coreceptor (38) Adult dorsal root ganglion neurons or postnatal cerebellar neurons from mice that are null for functional p75NTR are insensitive to MAG inhibition and Rho activation MAG does not appear to interact directly with p75NTR but could associate with it through GT1b (which specifically associates with p75NTR) Additionally, the newly discovered Nogo–NgR system also searches for a transmembrane signaling coreceptor Based on these leads, research has demonstrated that MAG, similarly to Nogo-66 (and OMgp), can bind NgR and engages p75NTR in neurite growth inhibitory signaling (24,25) Volume 34, 2006 If we have a direct link between MAG and p75NTR in the form of NgR, are the gangliosides still necessary? The answer appears to be affirmative, and therein is one major difference between MAG and the other NgR ligands Analysis of mice that were deficient in β1, 4-Nacetylgalactosaminyltransferase (ref 39; therefore lacking all complex gangliosides in the brain, including GD1a and GT1b) indicated that its neurons were no longer susceptible to the growth inhibitory effect of MAG-Fc but remained sensitive to Nogo-66 peptide (40) On the other hand, mice deficient in GD3 synthase (lacking the b-series gangliosides—that is, those with GD1a but not GT1b; ref 41) were still susceptible to both MAG-Fc and Nogo-66 Gangliosides are apparently also necessary for MAG’s activation of Rho Interestingly, both MAG-Fc and Nogo-66 peptide induce clustering of p75NTR into lipid rafts The latter appears to be required for the growth cone collapsing activity of myelin-associated inhibitors, because this activity is abolished by cholesterol extraction with methyl-β—cyclodextrin (40) It is clear from the aforementioned findings that gangliosides plays a role in modulating spatial, and perhaps temporal, response of axons to MAG in a way Nogo-66 is not subjected to How exactly does engagement of NgR and p75NTR by MAG activate Rho? In the cell, Rho is kept inactive in the cytosolic pool by its binding to the Rho GDP dissociation inhibitor (RhoGDI) (42) p75NTR–RhoGDI interaction is apparently strengthened by the binding of MAG or Nogo to the receptor complex, and therefore, p75NTR could activate Rho by somehow displacing it from RhoGDI A new insight to this theory has been obtained p75NTR was known to undergo ectodomain shedding and regulated intramembrane proteolysis, an αsecretase-mediated process that generates an extracellular domain and a C-terminal motif The latter is further cleaved by the γ-secretase complex to generate an intracellular domain (43,44) Filbin et al (45) have shown this PKCdependent cleavage of p75NTR by to be induced by MAG binding to cerebellar neurons, and it is necessary for Rho activation As Molecular Neurobiology Chen et al mentioned earlier, p75NTR activates Rho, presumably by acting as a displacement factor that realeases Rho from Rho-GDI (46) This finding has provided some possible resolution to the paradoxical observation that endogenous p75NTR is in complex with Rho–RhoGDI and that MAG/Nogo-66 binding appears to strengthen existing p75NTR–Rho–RhoGDI complexes rather than weaken it Therefore, upon cleavage the intracellular domain fragment of p75NTR, unlike full-length p75NTR, could perturb RhoGDI’s ability to inhibit GTP–GDP exchange on Rho, thus resulting in its dissociation from RhoGDI MAG’s Neuronal Interacting Partners II: Sialoglycoproteins Gangliosides are also not the only neuronal molecules that interact with MAG, because MAG-Fc binding to neurons is sensitive to trypsin (47), and a pull-down screen revealed several high-molecular-weight proteins interacting specifically with trypsin (48) Two NgR homologs (designated NgR2 and NgR3) were recently identified in the mammalian genome (49–51), but preliminary analyses provided the impression that these did not bind the known NgR ligands However, a more recent in-depth analysis revealed that MAG exhibits a sialic-aciddependent affinity for NgR2 that was severalfold higher than NgR (52) Interestingly, the study also revealed that MAG’s interaction with NgR is sialic-acid-dependent, whereas Nogo-66’s interaction with NgR was not Additionally, Nogo-66 does not interact with NgR2 Therefore, in addition to that observed for the sialoglycolipids, MAG and Nogo differ in terms of sialoglycoprotein binding Pertaining to MAG, NgR2 appears to have a role analogous to NgR because its ectopic overexpression in neurons confers susceptibility to inhibition by MAG However, it has not been determined whether this inhibition also occurs via the engagement of p75NTR/TAJ (and/or LINGO-1) as in the case of NgR Volume 34, 2006 MAG-Mediated Signaling in the CNS MAG Signaling and Axon–Glia Pathology MAG–ganglioside interaction goes beyond the signaling of neurite growth inhibition In fact, it has been proposed that at least some of MAG’s interaction with its neuronal ganglioside ligands is a lipid raft-to-lipid raft affair occurring on opposing oligodendroglia and axonal membranes (53) This implies a structural organization for bidirectional signaling that may be important for the stabilization of the axon–glia interaction Mice lacking complex gangliosides as a result of a deletion of GM2/GD2 synthase developed Wallerian degeneration, myelination defects, and, interestingly, a reduction in CNS MAG levels (54) Researchers recently reported that when backcrossed to a more than 99% C57BL/6 strain purity, MAG-deficient mice exhibited marked CNS (as well as PNS) axonal degeneration (55) that was qualitatively similar to mice deficient in β1, 4-N-acetylgalactosaminyltransferase (39) As mentioned earlier, MAG is apparently connected to oligodendrocyte differentiation and myelination via its interaction with Fyn kinase Therefore, a particular recent finding is noteworthy Mi et al (56) found that LINGO-1 is also expressed in oligodendrocytes and further showed that it appears to regulate myelination Accordingly, attenuation of LINGO-1 expression or activity in primary oligodendrocytes increases myelination competence, whereas the overexpression of LINGO-1 inhibits oligodendrocyte differentiation and myelination LINGO-1 affects the aforementioned processes via modulation of the expression and phosphorylation of Fyn, as well as the activation of Rho The exact mechanism by which this is achieved is unknown, as is how MAG could be involved in this connection On one hand, LINGO-1 action in oligodendrocytes may occur via pathways and signaling components independent of MAG until their convergence at Fyn and Rho However, although MAG’s interaction with the neuronal NgR complex containing LINGO-1 occurs in trans, it is not entirely inconceivable that MAG might interact in cis with a similar Molecular Neurobiology complex in oligodendrocytes and might modulate its activity somewhat These possibilities remain to be explored MAG and Other CCNS Neuropathology An interesting recent finding implicated MAG’s involvement, for the first time, in a human hereditary disorder: familial late-onset orthochromatic leukodystrophy (57) This genetic neurological disease has no clear signs of neuropathy Analysis of two members of an Italian family with the disease indicated their brain myelin contained a truncated from of LMAG that is about kDa shorter than wildtype The defect is not the result of a mutation in either the coding or untranslated region of the MAG gene, and the alteration in MAG is likely to be secondary to a yet unknown primary defect that resulted in their production with age It is unclear how this truncated form of L-MAG affects the CNS white matter It should be noted that unlike rodents, L-MAG is the predominating form in the adult human CNS Therefore, any alteration to the protein is likely to result in either a loss- or gain-of-function defect that could lead to oligodendroglial or neuronal pathology Another aspect of MAG that has been recently explored involves its use as a therapeutic target in CNS injuries Preclinical intervention models targeting the NgR with in situ delivery of dominant-negative NgR proteins showed significant benefits in optic nerve injury and stroke (58,59) However, a MAG-specific agent for CNS injury has not been developed and explored until recently There are indications that a MAG-specific agent may be beneficial in brain injuries such as stroke In the rat, it has been shown that L-MAG is re-expressed in oligodendrocyte cytoplasms in the white matter around experimental cerebral infarcts produced by middle cerebral artery occlusion (MCAO) (60) MAG expression is elevated in cortical lesions, and neuraminidase treatment of axotomized entorhino-hippocampal cultures Volume 34, 2006 Chen et al Fig A schematic diagram of MAG-associated forward and reverse signaling pathways and components in the axon–glial system Engagement of neuronal NgR–p75NTR and LINGO-1 by oligodendroglial MAG mediates neurite growth inhibition, as measured by growth inhibition assays, resulting in the activation of RhoA and suppression of Rac (not shown here) (29) The receptor complex presumably activates the trimeric G protein Gi and the downstream phospholipase C–protein kinase C (PKC)/Inositol 1,4,5-triphosphate (IP3) pathways (as depicted in ref 67) PKC is activated by both phospholipase C-generated diacylglycerol and the elevation of growth cone cytoplasmic Ca2+ (a result of IP3-induced Ca2+ release from internal stores) PKC appears to be important for Rho activation, because PKC inhibitors attenuate MAG’s ability to inhibit neurite growth (68) and may, in some cases, enhance neurite growth (67) It is unclear exactly how PKC activity might affect Rho activation (dotted lines), but intramembrane proteolysis of p75NTR by γ-secretase is apparently PKC-dependent (45) Upon cleavage, the intracellular domain of p75NTR displaces Rho-GDI from Rho, leading to its activation by guanine nucleotide exchange factors (not shown here) In growth cone turning assays, p75NTR and NgR are also responsible for the intracellular activation of Ca2+ that resulted in growth cone turning (25) However, the effect of MAG-induced Ca2+ on growth cone behavior is complex It may mediate growth cone repulsion or attraction, depending on the exact concentration of Ca2+ elicited (69) This effect is modulated by intracellular cyclic adenosine monophosphate (cAMP) (69,70), which is elevated by neurotrophin (N) signaling through Trk family receptors The cAMP elevation has been demonstrated to overcome MAG’s inhibition of neurite outgrowth (71–73) The modulatory effect of cAMP and protein kinase A in modulating neurite outgrowth has been extensively reviewed It is not described here, and the reader is referred to the excellent and in-depth discussion by Filbin (74) for more details Note that neurite growth inhibition and growth cone repulsion and attraction are experimental phenomena Different experimental paradigms have different emphasis regarding parameters measured On the whole, it appears that the same set of pathways and components are engaged, but there may be subtle differences Fyn tyrosine kinase is a key modulator of oligodendrocyte differentiation and interacts with MAG Crosslinking of oligodendroglial MAG by antibodies could result in Fyn phosphorylation Differentiation of oligodendrocytes could be triggered by engagement of the extracellular matrix via integrins, whose signaling via Fyn to Rho family GTPases regulates morphological differentiation (75) LINGO-1 on oligodendrocytes modulates its differentiation by activating Rho It is unclear whether MAG has a direct role in this effect of LINGO-1 potentiates axonal regeneration (61) Irving and colleagues (62) found that an anti-MAG monoclonal antibody that could neutralize the neuronal inhibitory effect of MAG could also protect oligodendrocytes from glutamate-mediated oxidative stress-induced cell death Importantly, this antibody showed significant beneficial effects in a rat model of MCAO Central and systemic administration of the antibody h after stroke induction significantly reduced infarct volume at d Neuroprotection was also associated with significant improvement in motor function This finding indicates the potential for the use of anti-MAG antibodies as therapeutic agents for the treatment of stroke An emerging aspect of myelin-associated proteins in CNS pathology has occurred in the area of schizophrenia and bipolar disorder, which have been associated with oligodendrocyte dysfunction (63,64) MAG has been shown to be Molecular Neurobiology downregulated in schizophrenic brains Genetic studies linking polymorphisms at the MAG locus to schizophrenia have also been reported recently for Chinese family cohorts (65,66) It remains to be seen whether these linkages are borne by more extensive studies, or whether the association between MAG and schizophrenia will be as controversial as that of Nogo Conclusion Although a major portion of MAG’s functional effect on neurons in CNS pathophysiology is likely to be mediated by NgR, MAG’s actions differ from that of Nogo-66, as shown by the availability of ganglioside coreceptors (see Fig for a detailed review of some of the signaling pathways and components associated with MAG) Neuronal receptors that are Volume 34, 2006 MAG-Mediated Signaling in the CNS MAG-specific, such as NgR2, also confer a unique neuronal response to MAG MAG clearly has other neuronal binding partners that have yet to be molecularly identified Molecular Neurobiology Some of these may modulate its interaction with the NgR complex or downstream signaling, whereas others may have completely unrelated effects MAG’s reverse signaling in Volume 34, 2006 oligodendrocytes has remained largely unexplored The availability of promising new molecular handles may soon change this Contra-indicative to the subtlety of its knockout phenotype, further investigations in MAG–neuron interaction and MAG reverse signaling and continuous exploration of its value as a therapeutic target in CNS pathophysiology now appear to be worthwhile ventures Acknowledgments Work on neuronal regeneration in B L Tang’s laboratory is supported by research grant number 03/1/21/19/247 from the Agency for Science, Technology, and Research (A*STAR)’s Biomedical Research council (BMRC) Chen et al 10 11 References Lai C., Brow M A., Nave K A., et al (1987) Two forms of 1B236/myelin-associated glycoprotein, a cell adhesion molecule for postnatal neural development, are produced by alternative splicing Proc Natl Acad Sci USA 84, 4337–4341 Pedraza L., Frey A B., Hempstead B L., Colman D R., and Salzer J L (1991) Differential expression of MAG isoforms during development J Neurosci Res 29, 141–148 Miescher G C., Lutzelschwab R., Erne B., Ferracin F., Huber S., and Steck A J (1997) Reciprocal expression of myelin-associated glycoprotein splice variants in the adult human peripheral and central nervous systems Mol Brain 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Expression of AMIGO in rodent brain 45 3.1.2 Expression of AMIGO in primary cultured neurons and glia cells .47 3.2 Polarized neuronal surface localization of AMIGO 55 3.3 The role of AMIGO