Elsevier

Brain, Behavior, and Immunity

Volume 24, Issue 8, November 2010, Pages 1254-1267
Brain, Behavior, and Immunity

Named Series: Biology of Microglia
Transcriptional profiling of the injured sciatic nerve of mice carrying the Wld(S) mutant gene: Identification of genes involved in neuroprotection, neuroinflammation, and nerve regeneration

https://doi.org/10.1016/j.bbi.2010.07.249Get rights and content

Abstract

Wallerian degeneration (WD) involves the fragmentation of axonal segments disconnected from their cell bodies, segmentation of the myelin sheath, and removal of debris by Schwann cells and immune cells. The removal and downregulation of myelin-associated inhibitors of axonal regeneration and synthesis of growth factors by these two cell types are critical responses to successful nerve repair. Here, we analyzed the transcriptome of the sciatic nerve of mice carrying the Wallerian degeneration slow (WldS) mutant gene, a gene that confers axonal protection in the distal stump after injury, therefore causing significant delays in WD, neuroinflammation, and axonal regeneration. Of the thousands of genes analyzed by microarray, 719 transcripts were differentially expressed between WldS and wild-type (wt) mice. Notably, the Nmnat1, a transcript contained within the sequence of the WldS gene, was upregulated by five to eightfold in the sciatic nerve of naive WldS mice compared with wt. The injured sciatic nerve of wt could be further distinguished from the one of WldS mice by the preferential upregulation of genes involved in axonal processes and plasticity (Chl1, Epha5, Gadd45b, Jun, Nav2, Nptx1, Nrcam, Ntm, Sema4f), inflammation and immunity (Arg1, Lgals3, Megf10, Panx1), growth factors/cytokines and their receptors (Clcf1, Fgf5, Gdnf, Gfrα1, Il7r, Lif, Ngfr/p75NTR, Shh), and cell adhesion and extracellular matrix (Adam8, Gpc1, Mmp9, Tnc). These results will help understand how the nervous and immune systems interact to modulate nerve repair, and identify the molecules that drive these responses.

Research highlights

► Gene profiling of the sciatic nerve of Wallerian degeneration slow (WldS) mutant mice. ► The WldS mutation causes delays in neuroinflammation and axonal regeneration. ► Genes that promote axon growth (Chl1, Clcf1, Fgf5, Gdnf, Jun, Lif, Nav2, Nrcam, Ntm, Shh) are downregulated in WldS mice. ► Injured WldS mice express more strongly axon growth inhibitory molecules found in myelin and scar tissue ► Genes associated with immunosuppression (e.g. Fpr2, Cd55, Cd59) are expressed at higher levels in mutants. ► IFN-α/ß signaling pathway could also contribute to immunosuppression in WldS mutants.

Introduction

Efforts aimed at promoting regeneration and repair of the injured adult central nervous system (CNS) involves trying to understand and recapitulate some of the events occurring in vivo in neural tissue capable of healing itself, such as the injured peripheral nerve. However, because of the complexity of the peripheral nervous system (PNS), especially in the context of an injury causing the activation, proliferation, recruitment, and differentiation of various cell types, it is difficult to disentangle the exact role of the hundreds of genes that are regulated and their relative importance to the regenerative repair process. An animal model that has been particularly useful for identifying novel candidate genes that may regulate neuroprotection and neurodegeneration in injury and disease has been the Wallerian degeneration slow (WldS) mutant mouse (Coleman, 2005, Coleman and Freeman, 2010).

The WldS mouse harbors a spontaneous mutation that results in the generation of a chimeric fusion protein that potently suppresses Wallerian degeneration (WD) after axotomy. In these mice, severed distal axons are protected from fragmentation, and immune cell recruitment and axonal regeneration are significantly delayed (for review, (Coleman and Freeman, 2010)). Interestingly, the Coleman laboratory has recently demonstrated the presence of the WldS mutant protein in axons in vivo (Beirowski et al., 2009), therefore suggesting that genes whose expression is upregulated or downregulated in the axon per se might be responsible for the WldS phenotype. The fact that WldS axons can survive for prolonged periods without their cell bodies and in the absence of both immune cells and glia further supports this possibility (Beirowski et al., 2004, Glass et al., 1993, Lunn et al., 1989, Perry et al., 1991, Perry et al., 1990). For these reasons, we decided to focus our attention on genes that are differentially regulated in the nerve distal stump, as opposed to where cell bodies of axotomized neurons are located (e.g. DRGs, spinal cord). Furthermore, analyzing gene expression profiles in the nerve distal stump offers the possibility to study the response of Schwann cells and immune cells to injury.

Section snippets

Animals

A total of 184 mice (8–12 weeks old) were used in this study. C57BL/6 (wild-type, wt; n = 92) and mutant C57BL/6 OlaHsd-WldS (WldS; n = 92) mice were purchased from Harlan UK Limited (Bicester, UK). Mice had ad libitum access to food and water.

Sciatic nerve lesions

Mice were deeply anesthetized with isoflurane and underwent a microcrush lesion of their left sciatic nerve at the mid-thigh level, following our previously published method (Boivin et al., 2007). As before, the lesion site was marked with a 10-0 Ethilon

Identification of genes that are differentially regulated in the sciatic nerve distal stump of WldS and wt mice after injury

Our goal was to identify genes that play important roles in neuroprotection, neuroinflammation, and nerve repair. To achieve this goal, we took advantage of microarray technology and mice harboring the WldS mutation that confers significant axon protection following axotomy, thereby causing concomitant delays in inflammation, axonal regeneration, and remyelination. Our working hypothesis was that genes that are preferentially upregulated in the sciatic nerve distal stump of wt mice compared to

Discussion

Since the unexpected discovery of the spontaneous mouse mutant WldS at Harlan-Olac (Bicester, UK) in the late 1980s (Lunn et al., 1989), a great number of studies have used this mouse model to investigate the mechanisms by which WldS delays axon degeneration following axotomy (for reviews, see (Coleman and Freeman, 2010, Vargas and Barres, 2007)). The WLDS protein has now been identified and its transgenic expression in various animal species conferred protection of transected axons (Adalbert

Acknowledgments

This study was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) to S.L. S.L. is supported by a Research Career Award from the Fonds de la recherche en santé du Québec (FRSQ). We are grateful to Nadia Fortin for her invaluable technical assistance. We thank the CHUL Research Centre’s Genomic Core Facility for assistance with microarray analysis. We would also like to thank Richard Poulin for his help editing this manuscript.

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    Present address: Department of Neurogenetics, Max Planck Institute of Experimental Medicine, Goettingen, Germany.

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