Neurotractin/kilon promotes neurite outgrowth and is expressed on reactive astrocytes after entorhinal cortex lesion

Abstract

The IgLON subgroup of the immunoglobulin superfamily consists of four members that are thought to be important in neural cell–cell recognition. Here, we cloned and characterized the murine IgLON subgroup member neurotractin/kilon, in the context of brain development and axonal regeneration. Neurotractin/kilon was found to be upregulated during brain development and is expressed on neurites of primary hippocampal neurons. To elucidate a potential role for neurotractin/kilon during regeneration in the CNS, we performed lesions in the entorhinal cortex, and showed that the expression of neurotractin/kilon is induced on reactive astrocytes. Notably, the expression on reactive astrocytes appears specifically in the denervated outer molecular layer of the dentate gyrus, where regenerative axon sprouting occurs. In vitro assays demonstrated that neurotractin/kilon attracts hippocampal axons in the stripe assay and that astroglial neurotractin/kilon promotes neurite outgrowth. These results suggest a function for neurotractin/kilon as a trans-neural growth-promoting factor for outgrowing axons following hippocampal denervation.

Introduction

A number of neurite-associated members of the immunoglobulin superfamily (IgSF) represent local cues for the development of neuronal circuits. This superfamily can be divided into several distinct structural subgroups, for example, the L1, the contactin/F11, or the robo subgroups (Brümmendorf and Rathjen, 1996, Kamiguchi and Lemmon, 2000, Tessier-Lavigne and Goodman, 1996). The recently characterized IgLON subgroup of the IgSF comprises four members: LAMP, OPCML/OBCAM, neurotrimin/CEPU-1, and neurotractin/kilon. These GPI-linked proteins consist of three Ig-like domains and localize to distinct but overlapping populations of neurons (Brümmendorf et al., 1997, Funatsu et al., 1999, Levitt, 1984, Marg et al., 1999, Schofield et al., 1989, Spaltmann and Brümmendorf, 1996, Struyk et al., 1995). Several in vitro studies showed that members of the IgLON subgroup act in various aspects of cell–cell recognition between neuronal subpopulations. For example, LAMP stimulates neurite outgrowth of limbic axons (Pimenta et al., 1995), modulates branching and layer specificity of thalamic axon systems (Mann et al., 1998) but blocks neurite outgrowth of dorsal root ganglia (DRG) neurons. In contrast, neurotrimin supports DRG neurite outgrowth but inhibits neurite extension of sympathetic neurons (Gil et al., 1998, Gil et al., 2002). Inhibition of neurite outgrowth was also observed for heterodimers of CEPU-1/neurotrimin and OBCAM using cerebellar granule cells (Reed et al., 2004). These data indicate a bifunctional activity for IgLON members in that they enhance or inhibit neurite extension. IgLON members are not restricted to the nervous system. Recently, OPCML, the human orthologue of OBCAM, has been identified as a tumor suppressor gene in epithelial ovarian cancer (Sellar et al., 2003). Loss of OPCML on these cells might reduce their intercellular adhesion, and thereby accelerates cell proliferation.

Here, we aimed to study the function of neurotractin/kilon in the mature brain, particularly in the context of axonal regeneration. We used entorhinal cortex lesions in mice as a model system for CNS regeneration in mammals. Interestingly, we found that neurotractin/kilon, which has been considered previously as a neuronal protein, is strongly upregulated on reactive astrocytes that are specifically located in denervated zones of the hippocampus after entorhinal cortex lesion. Thus, expression of neurotractin/kilon coincides with the onset of regenerative axon sprouting in this region (Frotscher et al., 1997, Matthews et al., 1976, Savaskan and Nitsch, 2001, Steward et al., 1988). Since neurotractin/kilon is known to be involved in neurite outgrowth of telencephalic neurons from embryonic chick brain (Marg et al., 1999), we analyzed the presumptive neurite outgrowth-promoting ability of mouse neurotractin/kilon in three different in vitro assay systems. These experiments revealed a neurite outgrowth-promoting function of mouse neurotractin/kilon on astrocytes for hippocampal neurons. Taken together, these results suggest a function for neurotractin/kilon as a trans-neural growth-promoting factor in regenerative axon sprouting in the adult mammalian brain.