Neurotractin/kilon promotes neurite outgrowth and is expressed on reactive astrocytes after entorhinal cortex lesion
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.
Section snippets
Neurotractin is developmentally upregulated in the CNS and is expressed on hippocampal neurites
Previously, we described avian neurotractin as a novel member of the IgSF subgroup IgLON, which is implicated in neurite formation in vitro (Marg et al., 1999). To study its function in the mammalian brain, we cloned neurotractin from a neonatal mouse brain cDNA library by PCR. Comparison of the deduced amino acid sequence revealed a 79.9 and 99.4% identity to chick neurotractin and to kilon, the rat orthologue of neurotractin (Fig. 1A) (Funatsu et al., 1999, Marg et al., 1999). In all three
Discussion
To understand the significance of the cell surface protein neurotractin for developmental and regenerative processes in the CNS, we analyzed the regulation of neurotractin expression during development and after brain lesion in mice. Neurotractin is widely localized in the mouse brain and expressed predominantly by neurons. Neurotractin expression gradually increases during postnatal development and persists from this stage on. Although IgLON members are localized in distinct but overlapping
Cloning of murine neurotractin cDNA
The cDNA of neurotractin was amplified by PCR from a neonatal mouse brain library (Stratagene), using the primers 5′-TATGTCGACAGCAGCCTGGCAGGCACG-3′, and 5′-TATGTCGACCGTACAGACGGCCGATTGTAT-3′. The PCR conditions were 94°C for 3 min, and 34 cycles of 94°C for 30 s, 65°C for 30 s, and 72°C for 2 min. The PCR product was cloned via SalI into the pBluescript KS vector (pBSKS, Stratagene) and confirmed by DNA sequencing (Invitek). The cDNA sequence for murine neurotractin is available from
Acknowledgments
We acknowledge the technical assistance of Dieter Jobsky, Füsun Usku and Bettina Brokowski and the critical reading of the manuscript by Dr. Robert Nitsch. We are grateful to Dr. Gianfranco Gennarini for providing contactin-1-specific antibodies. This study was supported by a grant of the Deutsche Forschungsgemeinschaft (DFG). N.E Savaskan is a fellow of the DFG grant 1041/4-1.
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2016, Biochimica et Biophysica Acta - Molecular Cell ResearchCitation Excerpt :Schafer et al. [3] also reported that after entorhinal cortex lesion, expression of NEGR1 protein is induced on reactive astrocytes, which in turn promotes neurite outgrowth of hippocampal neurons. This suggests a function for NEGR1 as a trans-neural growth-promoting factor for outgrowing axons following hippocampal denervation [3]. Furthermore, Negr1 mRNA expression is inversely regulated during ischemic injury as compared to neuronal development [7,8] indicating that inverse regulation of neuronal genes during disease could contribute to neuronal death.
- 1
Present address: Institute de Neurobiology de la méditerranée, Campus de Luminy, 13288 Marseilles, France.
- 2
Present address: Division of Cellular Biochemistry, The Netherlands Cancer Institute (NKI), 1066 CX Amsterdam, The Netherlands.
- 3
Present address: Novartis Institutes for Biomedical Research, CH-4002 Basel, Switzerland.