Astrocyte influences on oligodendrocyte progenitor migration

B. Castellano Ldpez and M. Nieto-Sampedro (Edr.) Pmgress in Brain Research, Vol. I32 0 2001 Elsevier Science B.V. All rights reserved

CHAPTER 9

Astrocyte influences on oligodendrocyte progenitor migration 0. Schniidelbach * and J.W. Fawcett Department of Physiology, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK

Introduction Oligodendrocyte cell migration is required for the development of the nervous system and is crucial to determine the places of myelination in the central nervous system. During migration, oligodendrocyte progenitors frequently come across astrocytic surfaces and astrocyte-derived matrix. We have investigated the role of the calcium-dependent adhesion molecules, the cadherins, in oligodendrocyteastrocyte interaction and oligodendrocyte progenitor migration. Immunostaining demonstrated the expression of N-cadherin on the surfaces of both oligodendrocytes and astrocytes, and oligodendrocyte-like cells adhered to and spread on N-cadherin substrates. The blocking of cadherin function by antisera or specific peptides reduced adhesion of oligodendroglia to astrocyte monolayers, diminished contact time between oligodendrocyte processes and individual astrocytes, and significantly increased the migration of oligodendrocyte-like cells on astrocyte monolayers. Furthermore, a soluble cadherin molecule without adhesive properties increased oligodendroglial proliferation on various extracellular matrix substrates. These data suggest that cadherins are at least partially responsible for the poor migration-promoting

* Corresponding author: Oliver Schnldelbach. Present address: Aventis Research and Technologies, Industriepark Hoechst, Geblude G 830,69526 Frankfurt, Germany. Tel.: +49-69-3051-3146; Fax: +49-69-305-8153; E-mail: [email protected]

properties of postnatal astrocytes and that decreasing cell-cell adhesion might be used to effect repopulation of demyelinated multiple sclerosis lesions by oligodendrocyte progenitors.

O ligodendrocyte motility is important for the architecture of the central nervous system Cells of the oligodendrocyte lineage must migrate considerable distances from their area of origin (for example, from the subventricular zone of the cerebellum and developing forebrain to their final destinations in future white matter areas) in order to fulfil their role as the myelinating cell type in the central nervous system (CNS). Similarly, in other areas of the brain (such as optic nerve or spinal cord), sites of oligodendroglial generation are frequently distant from their sites of myelination (Levison et al., 1993; Pringle and Richardson, 1993; Cameron-Curry and Le Douarin, 1995; Ben-Hur et al., 1998). Oligodendrocyte progenitors (OPs) encounter astrocytes as they migrate into the optic nerve and spinal cord white matter. Remyelination in demyelinating diseases, such as multiple sclerosis, also requires cell migration within the astrocytic scar environment that is found in plaques. In this context, embryonic astrocytes have been shown to promote OP migration, while postnatal astrocytes are moderately inhibitory (Fok-Seang et al., 1995). A variety of molecular determinants have been implicated in migration of OPs. Some regulatory cytokines and signalling molecules have been found to either promote or inhibit migration (Fok-Seang

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et al., 1998). Several proteases, which most likely alter matrix properties have also been found to affect m igration (Amberger et al., 1997; Muir et al., 1998; Uhm et al., 1998). Furthermore, adhesive or antiadhesive cell surface molecules (such as integrins and the recently discovered AN2 glycoprotein), extracellular matrix (ECM) glycoproteins (e.g. laminin, fibronectin and tenascin),proteoglycans,and sialylated carbohydrate structures found on the embryonic form of the neural cell adhesionmolecule N-CAM seemto be of crucial importance for OP m igration. (Milner and ffrenchConstant, 1994; Wang et al., 1994; Fok-Seang et al., 1995; Frost et al., 1996; M ilner et al., 1996; Schnadelbachand Faissner, 1996; Schnadelbachet al., 1998; Niehaus et al., 1999). Adhesion and cell m igration Cell m igration requires that the m igrating cell be motile. Motility is a product of the intrinsic properties of the cells and of the various signalling pathways within them, some of which may be affected by adhesionmolecules (McCarthy and Turley, 1993). M igration is also directly influenced by cell adhesion: cells must adhere to the surface in order to m igrate, but must also releasethese adhesivecontacts at the trailing edge of the cell in order to move ahead (Huttenlocher et al., 1995). Thus, differential levels of adhesion and differential responses from the m igrating cells seem to be necessary to allow maximum m igration in diverse cellular systems.Various experiments(DiMilla et al., 1993; Paleceket al., 1997) demonstratedthe requirement of intermediate levels of adhesion to effect maximum m igration of transfected CHO cells, with either too little or too much adhesion being inhibitory. Along these lines, both adhesive and anti-adhesive molecules can increasem igration (Huttenlocher et al., 1995). Cadherins and cell m igration The family of calcium-dependent adhesion molecules, known as the cadherins, have been demonstrated to be involved in various aspects of cellular m igration (Letourneau et al., 1990; Chen et al., 1997). For example,neural crest cells lose their N-cadhe-

tin content prior to acquiring m igratory status. N-Cadherin is reexpressedby these cells upon reaggregation into sensory ganglia (Hatta and Takeichi, 1986). Also, the loss of cadherin expressionhas been demonstratedto be necessaryfor the acquisition of motility in adult songbird forebrain neuronal precursor cells (Barami et al., 1994) and blocking cadherin function can increase the motility of tumour cells (Behrens et al., 1989). Apparently, the existence of cadherins in these systems confers a high degree of adhesion between the cells so that motility is impaired. However, during developmentof the nervous system, cadherins seem to play an important role in the development of the retina and the guidance of optic nerve fibres (Matsunagaet al., 1988) as well as promoting the m igration of sensory growth cones on Schwann cells (Letourneau et al., 1990). Recently, N-cadherin has been shown to be involved in the control of Schwanncell m igration, in that the blocking of cadherin adhesive interactions by antibodies or specific peptides m imicking the cell adhesionsite caused a significant increasein Schwann cell m igration on astrocytic monolayers (Wilby et al., 1999). Cadherin and oligodendrocytes Cadherins are expressed by most cell types of the CNS and in particular, oligodendrocytes have been shown to adhere to and spread on N-cadherin substrates (Tomaselli et al., 1988; Redies and Takeichi, 1993; Payne and Lemmon, 1993; Payne et al., 1996, Honjo et al., 2000). N-Cadherin immunostainingcan be found on the surface of perinatal astrocytes and may therefore play an important role in the control of oligodendrocyte adhesion and m igration. To clarify this issue, experimentswere carried out dealing with oligodendrocyte behaviour upon encountering N-cadherin molecules or astrocytic surfaces (Schnadelbachet al., 2000). When offered as a substrate,both primary oligodendrocytesand the immortalised oligodendrocyte cell line CG4 (Louis et al., 1992) quickly adheredto and spreadon this surface. This reaction could be perturbed by adding functionblocking agents,such as antiseraor specific peptides m imicking the cadherin adhesionsite with the amino acid sequenceHAV (Blaschuk et al., 1990). Similarly, these blocking agents reduced the adhesion of freshly subcultivatedoligodendrocytesto established

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astrocyte monolayers, whereas control peptides and antibodies did not. Video-microscopy of individual oligodendrocyte-astrocyte interactions revealed that the normally long-lasting interactions between these two cell types were significantly shortened when blocking agents were present in the culture. Similarly to other adhesion molecules like laminin and fibronectin, the adhesion force conveyed by N-cadherin might be suitable for OP migration. To test this, OPs were cultivated in high density on a glass cover-slip fragment, which was subsequently inverted onto a surface consisting of N-cadherinhuman Fc fusion protein immobilised by an anti-Fc antibody (Walsh et al., 1997). Migration is assessed by counting cells in relation to their distance from the coverslip edge (Fok-Seang et al., 1995). N-Cadherin as a substrate alone did not promote migration in this assay, and OPs cultivated on this substrate remained completely immobile. But did N-cadherin simply not promote migration or was it actively inhibitory? When the N-cadherin-Fc fusion was immobilised as a co-substrate with the migration-promoting adhesion molecule laminin, it was capable of reducing oligodendrocyte migration in a dose-dependent manner. This effect could be neutralised by adding the blocking HAV peptide to the culture, thereby restoring OP migration. Since postnatal astrocytes possess surface characteristics which are less than optimal for oligodendrocyte migration, we assumed that N-cadherin might be an important migration-limiting factor. Therefore, we used the established coverslip migration technique and inverted the coverslip onto an astrocytic monolayer. In this assay, migration of OPs and CG4 cells on astrocytes is limited and clearly much less than on a laminin substrate alone, although astrocytes are known to produce migration-promoting molecules (Fok-Seang et al., 1995). Consequently, the migration of CG4 cells on astrocytes was significantly increased when cadherin-blocking agents were present, both in terms of migration distance and percentage of migrating cells. When these two factors were taken together in a measurement of total migratory capability of the CG4 culture, this value could be increased by several hundred percent as compared to controls treated with control peptides (Schnadelbach et al., 2000).

Mechanisms of cadherin action Cadherins, like other adhesion molecules, e.g. members of the Ig-superfamily, have a modular structure. It is believed that homophilic adhesion between the classical cadherins is mediated by an adhesion site which contains the motif His-Ala-Val (HAV) in the first of five extracellular domains. The specificity of this motif, which is present in several different cadherins like N-, P- and E-cadherin seems to be determined by flanking amino acids, and artificial peptides resembling this structure have been used to inhibit cadherin adhesive function in a variety of cellular models (Newton et al., 1993; Overduin et al., 1995; Shapiro et al., 1995; Willems et al., 1995; Makrigiannakis et al., 1999; Noe et al., 1999; Williams et al., 2000). N-Cadherin is known to affect cell behaviour in several different ways, one of which is by binding to the intracellular signalling proteins, known as the catenins (Takeichi, 1991, Vleminckx and Kemler, 1999). The catenins link N-cadherin to the cytoskeleton. One of the catenins, known as b-catenin is also capable of translocating to the nucleus and affecting gene expression (Gumbiner, 1995). Cadherin-catenin association and function may be modulated by tyrosine phosphorylation of the catenins (Daniel and Reynolds, 1997; Levenberg et al., 1998) and the association of a protein tyrosine phosphatase with the cytoplasmic domain of N-cadherin has been correlated to the regulation of the linkage of cadherin to the cytoskeleton (Balsam0 et al., 1996; Balsam0 et al., 1998) and thus, cadherin function (Soler et al., 1998). Interestingly, in another model system of cellular migration, the neural crest, a signalling interaction between N-cadherin and integrins has been found. Blocking of bl and 83 integrin adhesion by RGD peptides caused signalling events involving Ca*+ influx in neural crest cells and catenin phosphorylation that controlled distribution and activity of N-cadherin, resulting in N-cadherin-mediated cell aggregation (Monier-Gavelle and Duband, 1997). Furthermore, N-cadherin can c&interact with the FGF-receptor and affect its signalling properties (Williams et al., 1994; Walsh et al., 1997). Basic FGF is known to increase proliferation of OPs (BBgler et al., 1990), and we have observed a mitogenic influ-

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ence of the above mentioned N-cadherin fusion protein on our CG4 cultures (Schnadelbachet al., 2000). It is interesting to note that in the development of the mouse brain, N-cadherin mRNA is downregulated from El6 onwards, only shortly after PDGFcl-receptor immunopositive OPs appear.Limited amounts of N-cadherin mRNA are retained up to P6, but in the adult mouse brain, N-cadherin mRNA is at low levels and cadherin protein is mainly found at synapses (Pringle and Richardson, 1993; Redies and Takeichi, 1993; Uchida et al., 1996; Benson and Tanaka, 1998). This time course coincides with the period of OP migration and initiation of myelination in the CNS. However, OP migration in the damaged adult CNS appearsto be limited to distances of about 2 mm, and N-cadherin might be responsible for this phenomenon. In the regenerating PNS, N-cadherin is upregulatedat the contact sites between Schwann cells and peripheral nerve axons (Shibuya et al., 1995), and it has been shown to be upregulated on protein levels in the glial scar in the rat CNS (Vazquez-Chonaand Geisert, 1999). Conclusions Cadherins are considered to be the most important adhesion molecules in tissue segregation (Takeichi, 1991, 1995). Its strong adhesive properties clearly make it a prime candidate for strong intercellular and intraorgan interactions. These properties, however, also seem to hinder the migration of cells in several model systems, among them the migration of OPs during CNS development and regeneration. In this respect, N-cadherin can indirectly influence myelination in that the position of the myelinating cell is altered through more or less migration when N-cadherin function is either blocked or introduced, e.g. through reexpression. Thus, if in demyelinating diseases, such as multiple sclerosis, N-cadherin is reexpressedin the same manner as in PNS and CNS injuries, the alteration of N-cadherin function might be a way of effecting remyelination. Abbreviations CNS OP ECM

central nervous system oligodendrocyte progenitors extracellular matrix

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