Elsevier

Experimental Neurology

Volume 201, Issue 2, October 2006, Pages 461-469
Experimental Neurology

Expression of nectin-1, nectin-3, N-cadherin, and NCAM in spinal motoneurons after sciatic nerve transection

https://doi.org/10.1016/j.expneurol.2006.04.026Get rights and content

Abstract

We here study the expression patterns of the cell adhesion molecules nectin-1, nectin-3, N-cadherin, and neural cell adhesion molecule (NCAM) in motoneurons after sciatic nerve transection (SNT). Nectins are a newly discovered family of adhesion molecules that colocalize with N-cadherin in synapses and are expressed in axons during development. By in situ hybridization (ISH), we found nectin-3, N-cadherin, and NCAM mRNA in uninjured motoneurons. In uninjured animals, nectin-3 mRNA was present in a few vesicular acetylcholine transporter (VAChT)-positive cells of small motoneuron size in lamina IX of the spinal cord. SNT induced a significant increase of nectin-1, nectin-3, and NCAM mRNA, but the signal for N-cadherin mRNA was not affected. After SNT, signal for nectin-3 mRNA appeared over most motoneurons. We next investigated the presence of N-cadherin and nectin protein in synapses on spinal motoneurons by immunohistochemistry. Only N-cadherin immunoreactivity was seen in close relation to synaptophysin staining, while nectin-1 and nectin-3 immunoreactivity did not display such proximity. SNT resulted in decreased immunoreactivity for N-cadherin around the motoneuron soma, while nectin-1 and nectin-3 immunoreactivity remained unchanged. In the peripheral sciatic nerve, nectin-3 immunoreactivity was observed both in controls and following injury and nectin-3 colocalized with both neurofilament and the Schwann cell marker S100. In addition, an increased ISH signal for nectin-3 mRNA could be seen over the proximal stump of the sciatic nerve after SNT. We conclude that motoneuron injury induces complex changes in the spatiotemporal expression pattern of the investigated cell adhesion molecules.

Introduction

Regeneration of spinal motoneurons is fundamental to functional recovery after peripheral nerve injury. The regenerative process includes interactions between the injured motoneuron and a variety of other cell types, both glia and neurons. The distal part of the damaged axon undergoes Wallerian degeneration while Schwann cells reorganize into the bands of Büngner. Interactions between axons and Schwann cells are mediated by cell–cell receptors such as N-cadherin and neural cell adhesion molecule (NCAM) (Martini and Schachner, 1988, Seilheimer et al., 1989). In the spinal cord, peripheral nerve lesion induces loss of synapses from the surface of motoneurons (Blinzinger and Kreutzberg, 1968, Brännström and Kellerth, 1998, Lindå et al., 2000). Therefore, cell adhesion molecules may also be of importance in mediating these processes around the motoneuron soma because such molecules serve as mediators of synaptic adhesion. Furthermore, astrocytes are activated after axotomy (Aldskogius et al., 1999, Graeber et al., 1988, Tetzlaff et al., 1988) and extend processes between the motoneuron surface and removed synapses (Chen, 1978). The cell adhesion molecules involved in these events are not known.

N-cadherin and NCAM have been extensively studied, also in the context of neuronal regeneration. N-cadherin is a classical cadherin, mediating Ca2+-dependent and homophilic adhesion (Fannon and Colman, 1996, Yamagata et al., 1995). N-cadherin is expressed by spinal motoneurons (Monks and Watson, 2001) and has been implicated in synapse formation (Benson and Tanaka, 1998, Jontes et al., 2004) and synaptic plasticity induced by injury (Brock et al., 2004, Shan et al., 2002). The molecule was recently shown to mediate specific interactions between photoreceptor axons and target neurons in Drosophila (Prakash et al., 2005). NCAM is probably important in axon guidance, since NCAM knockout mice lose the selective targeting of regenerating motoneurons (Franz et al., 2005). Increases in both N-cadherin and NCAM immunoreactivity occur in transected sciatic nerves (Thornton et al., 2005), but expression of the molecules in spinal motoneurons following this injury has not been addressed to our knowledge.

In addition to N-cadherin and NCAM, other cell adhesion molecules may be of importance in motoneuron regeneration. Nectins are a newly described family of surface receptors that has four members, nectin-1 through -4, each of which has two or three splicing variants (α, β, and γ). Nectins colocalize with N-cadherin in synapses in the hippocampus, with nectin-1 and -3 localized to the pre- and postsynaptic membrane, respectively (Mizoguchi et al., 2002). Nectins are Ca2+-independent adhesion molecules, with three immunoglobulin-like extracellular loops, a transmembrane region and a cytoplasmic domain. The nectins form both homo- and heterodimers (Satoh-Horikawa et al., 2000). The cytoplasmic domain of the nectins binds to afadin, an actin-filament binding protein (Sakisaka and Takai, 2004). Nectins also induce activation of Cdc42 and Rac small G proteins, leading to reorganization of the actin cytoskeleton and altered gene expression (Shimizu and Takai, 2003).

Some data exist on nectin distribution outside the brain. There is a high expression of nectin-1 protein in peripheral sensory neurons and a low expression in spinal motoneurons (Mata et al., 2001). Another study showed expression of nectin-1 mRNA in spinal motoneurons and could not exclude an additional glial expression (Haarr et al., 2001). The nectins have not been studied in neuronal regeneration but have been shown to mediate contacts between growing neurites and the commissural plate (Okabe et al., 2004), which indicates that nectins may play a role in axon guidance.

We here investigate nectin-1, nectin-3, N-cadherin, and NCAM in and around spinal motoneurons and in the peripheral nerve following sciatic nerve transection (SNT). We hypothesized that SNT induces changes in the expression patterns of these cell adhesion molecules, as the processes of synapse elimination, growth cone transformation, Schwann cell reorganization, and activation of glial cells involve complex alterations in cell–cell interactions. We focused on the first two weeks after axotomy since the cellular events described above all occur within this time frame.

Section snippets

Surgery

Female Sprague-Dawley rats (B & K Universal, Stockholm, Sweden) were anesthetized with chloral hydrate (40 mg/100 g body weight) and a 7–10 mm segment from the sciatic nerve was unilaterally resected below the obturator tendon. To compensate for loss of fluid and to provide analgesia, the rats were given a subcutaneous injection of saline (2 ml; 0.9 mg/ml NaCl) containing buprenorphine (0.05 mg/kg). During surgery, the rats were kept on a heating pad. After surgery, the animals were allowed to

mRNA levels in spinal motoneurons

We first used ISH to study mRNA levels for nectin-1, nectin-3, N-cadherin, and NCAM in spinal motoneurons. For nectin-1, no distinct accumulation of signal could be seen over uninjured motoneurons, but such an accumulation appeared after SNT (Figs. 1A, B). An accumulation of signal was clearly visible over uninjured motoneurons for both N-cadherin and NCAM (Figs. 1C, E). No difference could be seen in signal levels for N-cadherin after SNT (Fig. 1, Fig. 2), but for NCAM, a stronger signal was

Discussion

We investigated the expression of nectin-1, nectin-3, N-cadherin, and NCAM in motoneurons after SNT. To our knowledge, this is the first study addressing involvement of nectin adhesion molecules in the neuronal response to axotomy.

Most uninjured motoneurons did not display a distinct ISH signal for nectin-1 or nectin-3 mRNA. Interestingly, a few cells of motoneuron size in lamina IX of the spinal cord were clearly positive for nectin-3. These cells were somewhat smaller than the average alpha

Acknowledgments

This study was supported by The Swedish Research Council, Marcus and Amelia Wallenbergs minnesfond, Marianne and Marcus Wallenbergs stiftelse, and Karolinska Institutet.

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