N-cadherin differentially determines Schwann cell and olfactory ensheathing cell adhesion and migration responses upon contact with astrocytes

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Abstract

Olfactory ensheathing cells (OECs) and Schwann cells provide a cellular environment that promotes axonal outgrowth in several models of CNS injury. However, they exhibit different properties when in contact with astrocytes. Schwann cells, but not OECs, induce characteristics that typify hypertrophy in astrocytes and exhibit a poor capacity to migrate within astrocyte-rich areas, making them less favourable for transplant-mediated repair. N-cadherin has been implicated in the adhesion of Schwann cells to astrocytes. Despite indistinguishable expression of N-cadherin, Schwann cells adhered more strongly to an astrocyte monolayer and migrated more slowly on astrocytes when compared to OECs. We have examined the role of N-cadherin in mediating these cellular interactions using RNA interference and found differing effects. In Schwann cells, suppression of N-cadherin reduced heterotypic and homotypic adhesion and they gained adhesion properties more akin to OECs. In contrast, suppression of N-cadherin in OECs had no effect. These findings imply that N-cadherin is differentially regulated in OECs and Schwann cells.

Introduction

In recent years, olfactory ensheathing cells (OECs) and Schwann cells have been proposed as candidates for transplant-mediated repair of CNS lesions (Franklin and Barnett, 2000, Raisman, 2001, Wewetzer et al., 2002). Both cell types have a similar repair repertoire, being able to myelinate experimentally created demyelinated lesions (Barnett et al., 2000, Franklin et al., 1996, Imaizumi et al., 2000), and permit rapid saltatory conduction to take place (Imaizumi et al., 2000, Honmou et al., 1996) using similar signalling mechanisms (Smith et al., 2001). In addition, both OECs and Schwann cells can support axonal outgrowth in experimental models of spinal cord injury (Ramon-Cueto and Nieto-Sampedro, 1994, Ramon-Cueto et al., 2000, Li et al., 1997, Li et al., 2003a, Li et al., 2003b).

Despite these similarities, there are significant differences between the two cell types that may affect their candidature for transplant-mediated repair. For example, Schwann cells may have a limited potential to repair CNS damage due to their inability to migrate and survive within the CNS environment after transplantation (Baron von Evercooren et al., 1992, Franklin and Blakemore, 1993, Iwashita et al., 2000). This inhibitory environment is characterised by the presence of reactive astrocytes (Eng and Ghirnikar, 1994, Norenberg, 1994), which may be a restrictive component of CNS tissue since remyelination by Schwann cells is only observed in astrocyte-free areas (Shields et al., 2000, Woodruff and Franklin, 1999). This propensity of Schwann cells to avoid astrocyte-rich areas was also demonstrated in vitro where Schwann cells cultured with astrocytes formed discrete boundaries being unable to migrate amongst them (Ghirnikar and Eng, 1994, Wilby et al., 1999).

In contrast to Schwann cells, OECs interact much more favourably with astrocytes and are able to intermingle and migrate amongst co-cultured astrocytes (Lakatos et al., 2000), perhaps due to their inherent ability to exist in situ alongside astrocytes within the olfactory system (Doucette, 1991, Doucette, 1993). Moreover, another difference between Schwann cells and OECs is seen in the hypertrophic response elicited in co-cultured astrocytes when in contact with Schwann cells. In this case, astrocytes became enlarged and exhibit an enhanced expression of the hypertrophic marker glial fibrillary acidic protein (GFAP) and chondroitin sulphate proteoglycans (CS-PGs), an inhibitory component of the glial scar (Norenberg, 1994). Neither of these markers is up-regulated in similar OEC/astrocyte co-cultures, which may be an important consideration when trying to minimise glial scarring. Support for this recently came from in vivo studies demonstrating that transplanted Schwann cells within the CNS induced a greater host tissue inhibitory response to axon regeneration by up-regulating expression of CS-PG and astrocytic GFAP compared to that associated with OEC transplantation (Lakatos et al., 2003, Plant et al., 2001).

It has been suggested that the restriction of Schwann cell migration into astrocyte-rich regions may be mediated by contact inhibition, perhaps via N-cadherin (Wilby et al., 1999). N-cadherin is the predominant cadherin in the nervous system, and plays an important role in axonal guidance (Ranscht, 2000) and synapse formation (Bruses, 2000). Since OECs migrate more extensively within an astrocyte environment than Schwann cells, it was originally suggested that OECs may lack N-cadherin, though this does not seem to be the case (Lakatos et al., 2000).

Thus, the role of N-cadherin requires further investigation. In this study, we address by precise molecular intervention whether (i) N-cadherin is present and functions similarly in both OECs and Schwann cells and (ii) if silencing N-cadherin using RNA interference has similar, or distinct, biological effects on the interactions of the two glial cells with astrocytes in vitro.

Section snippets

Results

Previous in vitro assays using OECs and Schwann cells co-cultured with astrocytes highlighted differences in their ability to interact with astrocytes. We have confirmed the reproducibility of the confrontation assay (Lakatos et al., 2000, Wilby et al., 1999) and demonstrated that Schwann cells were less able than OECs to mingle with astrocytes and induced characteristics typifying hypertrophy (Fig. 1A). This phenomenon reflects the in vivo observations following transplantation of OECs and

Discussion

Although Schwann cells and OECs are derived from developmentally distinct regions, the neural crest and olfactory placode, respectively, they share many properties in common. These include similar antigenic and morphological characteristics (Franklin and Barnett, 2000, Ramon-Cueto and Valverde, 1995), growth factor requirements in vitro (Pollock et al., 1999), and their shared ability to promote axonal outgrowth and myelination in CNS lesions in vivo (Wewetzer et al., 2002) and in vitro (Devon

Cell cultures

Astrocyte cultures were prepared from cerebral cortices of 1–2-day-old Sprague–Dawley rat pups (Harlan Olac Ltd, Oxon, UK) as described previously (Noble and Murray, 1984). Cells were then maintained in DMEM (Gibco, Life Sciences, Paisley, Scotland) containing 10% foetal bovine serum (DMEM-FBS; FBS, Imperial Laboratories) on poly-l-lysine (PLL, 13.3 μg/ml; Sigma, Dorset, UK) coated flasks. These cells were used to study the interactions with Schwann cells and OECs and also to generate astrocyte

Acknowledgments

This work was supported by the International Spinal Research Trust (RF). SB is a Multiple Sclerosis Society Senior Research Fellow. We would like to thank Dr. Val Brunton for advice, Mr. Tom Glibey for FACS sorting, and Professor Pat Doherty for the gift of the N-cadherin peptide inhibitor.

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