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Two novel mammalian nogo receptor homologs differentially expressed in the central and peripheral nervous systems☆

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Abstract

The regenerative capacity of the adult mammalian central nervous system is restricted by the myelinating oligodendrocytes that form a nonpermissive environment for axonal growth. Currently only the Nogo receptor (NgR), in complex with p75NTR neurotrophin receptor is known to be involved in this inhibitory signalling in neurons. NgR is a common receptor for the three inhibitory myelin proteins Nogo-A, OMgp, and MAG. Here we describe two novel Nogo receptor gene homologs named NGRL2 and NGRL3 from human and mouse that, like NGR, encode putative leucine-rich repeat containing GPI-anchored proteins. We show by in situ hybridisation and by RT-PCR that NGRL mRNAs are predominantly expressed in the neurons of the embryonic and adult central and peripheral nervous systems, and that they together with NGR possess distinct and partially nonoverlapping expression patterns. We also show that all four members of the reticulon family, including Nogo-A, are widely expressed in the nervous system, and therefore are possible ligands for the NgRLs.

Introduction

The regenerative capacity of the adult mammalian central nervous system (CNS) is much compromised when compared to the embryonic CNS, to the peripheral nervous system (PNS), and to many other tissues (for review, see (Schwab, 2002). Indeed, because serious spinal trauma and stroke usually lead to permanent disability, much effort has been put on elucidating the molecular mechanisms that limit the regenerative potential of the CNS. Damaged nerve fibers in the CNS often undergo a furious process of regenerative sprouting that, however, is ultimately abortive, and fails to lead to a functional recovery as first noted in 1911 (Ramon y Cajal, 1991). A series of experiments have demonstrated that this regenerative failure is mainly caused by the nonpermissive environment formed by the myelinating oligodendrocytes. This is in striking contrast to axonal growth-promoting milieu generated by the Schwann cells in the PNS. Namely, injured CNS neurons can extend neurites into peripheral nerve grafts as first shown by David and Aguayo (1981). In addition, the emergence of CNS myelination coincides with the loss of neuronal regenerative capacity (Kapfhammer and Schwab, 1994) and suppression of the onset of myelination has been shown to extend the permissive period for the functional repair in spinal cord Savio and Schwab, 1990, Keirstead et al., 1992. The physiological reason for this poor regenerative ability of the CNS neurons might be a compromise with the ultimate need to maintain precise functional neuronal connections.

In recent years, three protein components of the oligodendrocyte myelin have been demonstrated to act as tonic inhibitors of axon regeneration in the CNS, namely MAG (myelin-associated glycoprotein) Mukhopadhyay et al., 1994, McKerracher et al., 1994, OMgp (oligodendrocyte-myelin glycoprotein) (Wang et al., 2002b), and Nogo-A GrandPre et al., 2000, Chen et al., 2000, Prinjha et al., 2000. In a surprising convergence, all three proteins were shown to bind to the receptor named Nogo receptor (NgR) expressed on the surface of neurons Fournier et al., 2001, Liu et al., 2002, Wang et al., 2002b, Domeniconi et al., 2002. As NgR is a glycosylphosphatidylinositol (GPI)-linked membrane protein and thus lacks the intracellular part, it was reasonable to suggest that it has a coreceptor that further conveys the oligodendrocyte-to-neuron inhibitory signalling. Recently this coreceptor was identified as p75NTR Wang et al., 2002a, Wong et al., 2002, a receptor well characterized for its roles in neurotrophin signalling (Dechant and Barde, 2002). Intracellularly, the Rho family of GTPases plays key roles in regulating neurite outgrowth by coordinanting the cytoskeletal actin rearrangements. Interestingly, neurotrophin signalling via p75NTR modulates Rho activity and thereby axonal outgrowth (Yamashita et al., 1999).

Whereas OMgp and MAG have no close homologs in the human genome, Nogo-A belongs to a four-membered family of reticulons. Nogo-A is one of the several transcripts generated from reticulon 4 gene (RTN4) by alternative splicing and promotor usage (Oertle et al., 2003a). Nogo-A transcript encodes a protein with a unique N-terminus and a C-terminal conserved 66-amino acid segment (known as Nogo-66) that is flanked by two putative transmembrane regions. The topology of Nogo-A protein remains under debate (Oertle and Schwab, 2003). In addition to the N-terminal part of Nogo-A, Nogo-66 mediates the inhibitory effects of Nogo-A on axon regeneration. A general feature for all reticulons is the presence of a homologous 66-amino acid loop, the reticulon domain. The functions of other members of the reticulon family in axon regeneration remain unknown, but reticulons have been suggested to play a general role in cellular physiology (Oertle and Schwab, 2003).

The expression patterns of NGR and RTN4 mRNAs and proteins have been studied in detail Fournier et al., 2001, Josephson et al., 2001, Josephson et al., 2002, Hunt et al., 2002, Huber et al., 2002. NgR is located on the plasma membrane of neurons, whereas Nogo-A is expressed both in neurons and in oligodendrocytes. In oligodendrocytes, Nogo-A protein is localized in the innermost adaxonal and outermost myelin membranes in apposition to NgR positive neurons Wang et al., 2002c, Huber et al., 2002. In neurons, Nogo-A protein is located in growing axons (Tozaki et al., 2002), proximal dendrites, along the cell membrane, and in rough endoplasmic reticulum (Jin et al., 2003). These studies on the expression of Nogo-A have challenged the model of glial-to-neuron inhibition as a sole function of the Nogo-A-NgR signalling pathway. Interestingly, the expression of NGR mRNA has been found not to be pan-neuronal in the CNS. For example, neurons in certain regions of basal ganglia and hypothalamus do not express NGR or express it at very low levels, and it is unclear what receptors mediate the inhibition of axon regeneration in these cell populations. Interestingly, expression patterns of NGR and Nogo-A mRNAs overlap only partially Hunt et al., 2002, Josephson et al., 2001, further suggesting that other receptors could mediate the effects of Nogo-A in NgR negative cells, and/or that Nogo-A has additional functions.

Duplication of chromosomal segments and individual genes in vertebrate lineage has probably enabled the duplicated genes to obtain new functions, and has also stabilized the homeostatic mechanisms by providing functional redundancy within gene families Dehal et al., 2002, McLysaght et al., 2002. Therefore, and because of the imminent therapeutic potential of strategies interfering with the NgR signalling in the settings of CNS trauma (GrandPre et al., 2002), we decided to search NGR homologs in vertebrate genomes. Here we characterize two novel NGR homologs named NGRL2 and NGRL3 (Nogo receptor like) from human and mouse genomes, and demonstrate that they are differentially expressed in the developing and adult CNS and PNS. We also show that all four reticulons, the putative ligands for the NgRLs, are widely but differentially expressed in the mouse nervous system. Additionally, we analysed the evolutionary origin of the NGRLs, and we propose that they emerged in an interchromosomal duplication event in the vertebrate lineage that also created POV2, a novel gene homologous to POV1, prostate cancer overexpressed gene 1.

Section snippets

Human and mouse NGR, NGRL2, and NGRL3 define a novel gene family encoding GPI-linked LRR proteins

Using bioinformatics, we identified two novel genes encoding proteins that share significant sequence similarity with NgR, and furthermore possess an identical composition of structural motifs with NgR. cDNAs covering the full-length protein-coding regions were identified by analysing overlapping expressed sequence tags (ESTs). Sequences of human and mouse NGRL cDNAs encoding full-length proteins cloned from brain RNA by RT-PCR were identical with the sequences predicted by bioinformatics. In

Discussion

In this study, we describe two novel genes named NGRL2 and NGRL3 that, like NGR, encode leucine-rich repeat-containing GPI-linked membrane proteins. We show that NGRL mRNAs are expressed in the neurons with specific expression patterns in the brain, spinal cord, and peripheral sensory and sympathetic ganglia, and that their expression is developmentally regulated. NgR and NgRLs are more closely related to each other than to other LRR proteins, and thus they form a new LRR protein family.

The

Cloning and expression analyses of the NGRLs

Partial EST and genomic sequences for NGRLs were identified with BLAST on the basis of their close homology with NGR. The full-length protein-coding sequences were acquired by analysing overlapping EST clones. Primers were designed to isolate human and mouse NGRL2 and NGRL3 cDNAs encoding full-length proteins. Total mouse brain RNA was purified with RNAwiz (Ambion, TX) as recommended by the manufacturer. Human RNAs were obtained from Clontech, CA. Superscript II reverse transcriptase (RT)

Acknowledgements

We thank Svetlana Vasilieva for technical expertise and help. This work was supported by the Academy of Finland, Sigrid Jusélius Foundation, and by funds from the Institute of Biotechnology, University of Helsinki.

References (44)

  • T Oertle et al.

    Nogo and its paRTNers

    Trends Cell Biol.

    (2003)
  • T Oertle et al.

    Genomic structure and functional characterisation of the promoters of human and mouse nogo/rtn4

    J. Mol. Biol.

    (2003)
  • W.D Schubert et al.

    Structure of internalin, a major invasion protein of Listeria monocytogenes, in complex with its human receptor E-cadherin

    Cell

    (2002)
  • H Tozaki et al.

    Expression of Nogo protein by growing axons in the developing nervous system

    Brain Res. Mol. Brain Res.

    (2002)
  • T Yamashita et al.

    Neurotrophin binding to the p75 receptor modulates Rho activity and axonal outgrowth

    Neuron

    (1999)
  • M.S Chen et al.

    Nogo-A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1

    Nature

    (2000)
  • S David et al.

    Axonal elongation into peripheral nervous system “bridges” after central nervous system injury in adult rats

    Science

    (1981)
  • G Dechant et al.

    The neurotrophin receptor p75(NTR)novel functions and implications for diseases of the nervous system

    Nat. Neurosci.

    (2002)
  • P Dehal et al.

    The draft genome of Ciona intestinalisinsights into chordate and vertebrate origins

    Science

    (2002)
  • A.E Fournier et al.

    Identification of a receptor mediating Nogo-66 inhibition of axonal regeneration

    Nature

    (2001)
  • A.E Fournier et al.

    Truncated soluble Nogo receptor binds Nogo-66 and blocks inhibition of axon growth by myelin

    J. Neurosci.

    (2002)
  • T GrandPre et al.

    Identification of the Nogo inhibitor of axon regeneration as a Reticulon protein

    Nature

    (2000)
  • Cited by (0)

    Sequence data from this article have been deposited with the DDBJ/EMBL/GenBank data libraries under accession Nos. AY250219 (human NGRL2), AY250221 (human NGRL3), AY250218 (mouse Ngrl2), and AY250220 (mouse Ngrl3).

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