Functional role of a glycolipid in directional movements of neurons ∗

Migration of neurons from their site of origin to their final destination is a critical and universal step in the formation of the complex structure of the nervous system. The migratory process is thought to be governed in part by genetically and epigenetically defined sequences of signals which are interpreted by migrating cells. The molecular mechanisms that underlie neuronal migration have been the subject of intense investigation. As in other developmental processes, many molecules must participate in neuronal migration. Some molecules, such as cell adhesion molecules and motor proteins, may contribute to discrete steps in the migration act; others, like extracellular signaling molecules, may regulate the activation and/or termination of the migration program. In this article we review findings from our group that demonstrate the functional role(s) of a specific glycolipid in neuronal migration and neurite outgrowth in the developing and adult nervous system.


INTRODUCTION
In the developing central nervous system (CNS) most neuronal precursors are generated in specialized germinal zones adjacent to the ventricle, termed the ventricular zone.A wave of secondary neurogenesis occurs late in development, often in the perinatal period, and produces huge numbers of interneurons.Three primary sites of secondary neurogenesis have been described: the external germinal layer (EGL) of the cerebellum, the dentate gyrus, and the subventricular zone (SVZ) in the telencephalon.Both the dentate gyrus and the SVZ maintain a proliferative population of stem cells throughout life and give origin to neurons destined for the hippocampal formation and olfactory bulb respectively (Altman 1969a,b, Gage et al. 1998, Lois et al. 1996, Luskin 1993).
In general, after terminal mitosis, immature neurons migrate within the neuroepithelium until they reach the various structural primordia where subsequent maturation and final differentiation occurs.Two general modes of migration have previously been defined in the developing nervous system: radial and tangential neuronal migration.Radial migration involves movement of the neuron orthogonally to the pia surface and most of the neurons migrate associated to a special glia, the radial glia.Guidance along radial glia is a common mechanism for the formation of laminated structures, such as the cerebral cortex and the cerebellum (reviewed by Hatten 1999).This migration process is a complex developmental program involving a series of steps, including cell-cell recognition, cell-cell adhesion, cell motility, and finally detachment from the glial fibers once neurons reach their destination (Rakic et al. 1994).
Glial-directed neuronal migration has been well studied, but relatively less is known about a distinct mode of migration that occurs tangential to the pial surface.Tangential migration is a prominent developmental feature in both the cerebellum and the olfactory bulb (Luskin 1993, Luskin and Boone 1994, Lois et al. 1996), but has also been described in other regions as for instance the migration of the luteinizing hormone-releasing hormone (LHRH-) positive olfactory placode cells (Norgren and Lehman 1991), cortical interneurons (De Carlos et al. 1996, Anderson et al. 1997, Tamamaki et al. 1997), and neurons in the cerebral cortical subventricular zone (O'Rourke et al. 1997).In the cerebellum, neuronal precursors move tangentially from the rhombic lip to form the external granular layer.As the olfactory bulb forms, future interneurons are generated in the telencephalic SVZ and migrate into the bulb in a long tangential pathway.It has been shown that glial cells do not guide this class of neuronal migration.Rather, in the EGL it has been proposed that neuronal precursors migrate associated with axons (Hynes et al. 1986) and in the SVZ neurons move rapidly along one another in unique chain formations independent of radial glia or axonal processes, in a migration that persists into adulthood (Wichterle et al. 1997, Doetsch et al. 1997).Molecular studies have shown that the mechanism of migration involves the sialylated form of NCAM (neural cell adhesion molecule) and is guided, in part, by negative chemotropism (Hu et al. 1996).

GANGLIOSIDES AND CELL MIGRATION
In our laboratory we are interested in the study of molecules involved in neuronal migration and neu-rite outgrowth.In our studies we have identified a particular glycolipid, 9-O-acetyl GD3 recognized by a monoclonal antibody (Constantine-Paton et al. 1986) that is involved in neuronal migration and neurite outgrowth.
Results of numerous investigations of cell motility and axonal sprouting have suggested that gangliosides serve an important function in cell substratum interactions and neurite extension.Studies of these cell surface, sialic acid-bearing lipids have, in general, lagged behind those on similarly distributed proteins because of technical difficulties in their isolation and identification.Nevertheless, monoclonal antibodies directed against the carbohydrate moieties of gangliosides have become increasingly important aids in dissecting the biological function of these molecules.In our own earlier studies we described the monoclonal antibody JONES which recognizes a subset of acetylated gangliosides in the developing rat brain (Constantine-Paton et al. 1986).These acetylated gangliosides are associated with cell migration and axon elongation throughout the brain (Mendez-Otero et al. 1988, Schlosshauer et al. 1988, Mendez-Otero and Constantine-Paton 1990, Mendez-Otero and Ramón-Cueto 1994).
To better understand the role of these gangliosides in neuronal migration we have used the developing rat cerebellum as a model system.The development of the cerebellum is an example of a situation in which cell migration clearly plays a crucial role in the final architecture and, presumably, therefore in the physiology of the system.There are several waves of migration in this region.Prenatally, Purkinje cells, Golgi cells, and deep nuclear neurons originate in the ventricular zone and migrate into the body of the cerebellar anlage.This is a radial migration and quite likely takes place on radial glial cells although there is no direct evidence for this mechanism in this case.The deep nuclear neurons subsequently migrate away from the Purkinje cells to their final locations.Meanwhile, a second migration begins.A new stem cell layer known as the external granule (or germinal) layer (EGL) orig-inates in the ventricular region at the caudal margin of the fourth ventricle (the rhombic lip).From here, the cells migrate over the outer (dorsal) surface of the developing cerebellum and, by the time of birth, have come to cover this surface completely (Altman 1969b, Altman andBayer 1985 a, b, c).Postnatally this EGL gives rise to several types of neurons, but predominantly to granule cells, which make up 90% of the neurons of the cerebellum.These granule cells arise from the mitotic external granular cells and migrate inward from the pial surface past the layer of Purkinje cells and into the future internal granular cell layer (Altman 1972a, b, 1982, Rakic 1971, 1981).This postnatal migration of postmitotic but undifferentiated granule cells has been shown to follow the process of Bergmann glia, a particular type of radial glia found in the cerebellum (Rakic 1971(Rakic ,1981)).The expression of 9-O-acetyl GD3 correlates particularly with regions and times of cell migration in the rat cerebellum.In the embryo (E14-18) the labeling is present extending from the ventricular to the pial surface of the cerebellar anlage and the immunoreactivity is distributed in a radially oriented pattern.Immunoreactivity is also present in the subpial region of the developing EGL, which, at this stage, begins its migration from the rhombic lip and is present as a densely packed zone of cell bodies in the caudal region of the anlage (Mendez-Otero et al. 1988).During the first two postnatal weeks the immunoreactivity for 9-O-acetyl GD3 is detected throughout the folium with a radially oriented pattern extending from the EGL and through the presumptive molecular layer corresponding to the migration of granule cells associated with the Bergmann fibers.The pattern of expression of 9-O-acetyl gangliosides in the developing cerebellum indicates that these molecules may play a role in neuronal migration in vivo.
To test this hypothesis we have investigated the expression of 9-O-acetyl gangliosides in two different types of cerebellar cultures prepared from dissociated postnatal rat cerebella.In the first type, cells are plated after dissociation under conditions where most of the glial cells develop a stellate morphology that anchors neurons but does not support their migration.In the second type of culture, cells are plated in a ratio of four neurons to one glia cell and under these conditions the predominant form of astroglia is an elongated form that supports the migration of granule neurons.Granule neurons express the gangliosides in both types of cultures.However, in cultures where the astroglial cells display the stellate morphology only a small proportion shows staining with the gangliosides.Cultures in which the glial cells assume the elongate morphology (radial glia) have a significantly higher number of Jonespositive astroglia (Mendez-Otero and Constantine-Paton 1990).We have also used cerebellar explants and slices from postnatal rat cerebellum in antibody perturbation assays to investigate the functional role of these ganglioside in granule cell migration.We found that although other receptor systems can support neuronal movement, blocking 9-O-acetyl GD3 reduces the rate of neuronal locomotion by approximately 66% (Santiago et al. 2001).
The migration of granule cells along the Bergmann glial fibers has been a useful model system to study radial neuronal migration.In rodents, this migration takes place during the first two postnatal weeks.These studies have implicated a number of molecules in steps of the migration process, for example, cell adhesion molecules, i.e., AMOG (Antonicek et al. 1987, Gloor et al. 1990) and astrotactin (Zheng et al. 1996); ion channels, i.e., NMDA receptors (Komuro and Rakic 1993) and Ca 2+ channels (Komuro and Rakic 1992).In addition, studies of mutant mouse lines with defects in brain development have led to the identification of other molecules, including potassium channels in the case of weaver mutant (Patil et al. 1995); the extracellular matrix protein reelin in the reeler mutant (Caviness and Sidman 1973, D'Arcangelo et al. 1995, Hirotsune et al. 1995); p35, an activator of Cdk5 (Chae et al. 1997); and neuregulin and erbB receptors (Rio et al. 1997).
Our finding that 9-O-acetyl gangliosides play a critical role in mediating neuronal migration in the cerebellum may also be relevant to neuronal migration in other regions of the developing nervous system.In this respect, we have shown that the expression of these gangliosides is not restricted to regions of radial neuronal migration but is also present in regions were tangential migration is taking place as for instance the EGL and the SVZ (Mendez-Otero et al. 1988, Mendez-Otero andCavalcante 1996).
Long tangential migrations are a prominent developmental feature in both the EGL and SVZ.It has been shown that glial cells do not guide this class of neuronal migration.Hynes et al. (1986) have suggested that in the prenatal migration en masse of EGL cells these cells migrate in close contact with axons that are present prior to the onset of migration and it appears that these axons serve as the substrate for EGL neuroblast migration and that axonal guidance of cell migration may be a general mechanism to be added to the previously studied guidance by glial cells processes.
We have investigated whether 9-O-acetyl gangliosides are also involved in tangential neuronal migration using the migration of the SVZ cells as a model.We have found that 9-O-acetyl gangliosides are highly expressed in the SVZ and along the route of tangential migration into the olfactory bulb during development (Mendez-Otero andCavalcante 1996, Miyakoshi et al. 2001).Furthermore, the expression of these gangliosides persists in adult in cell chains similar to those showing immunoreactivity in developing animals (Mendez-Otero and Cavalcante 1996).

NEURONAL MIGRATION AND NEURITE OUTGROWTH
Cell migration is a ubiquitous event that occurs in all multicellular organisms to different extents and at different stages in their lifetime.Cell movements occur not only during development but also during wound healing, immune response, and tumor formation.The idea that axon extension is a form of cell migration in which the cell soma remains sta-tionary was proposed many years ago (reviewed by Singer and Kupfer 1986) and has garnered support from findings that molecules that guide axons are also involved in cell migration.
We have investigated the role of 9-O-acetyl gangliosides identified by the Jones mAb in the elongation of neurites extended by neurons of embryonic rat dorsal root ganglia (DRG) explants growth in vitro.The behavior of individual growth cones was recorded using a time-lapse video-enhanced imaging system before and after the addition of antibodies that recognize specific gangliosides known to be expressed on these growth cones.It was possible to demonstrate that the advance of growth cones on laminin was halted in the presence of Jones mAb.This effect was partially reverted by washing the explants for several minutes with culture medium.Our findings show that 9-O-acetyl gangliosides may play an important role on the extension of growth cones and consequently influences navigation and pathway finding during development (Mendez-Otero andFriedman 1996, Araujo et al. 1997).

CONCLUSIONS AND PERSPECTIVES
The mechanisms by which ganglioside expression leads to neuronal migration and neurite outgrowth are still not defined.However, based on the data summarized in the previous sections and also in the literature (Cheresh 1987, Pande 2000, Armulik et al. 1999, Iwabuchi et al. 1998, Probstmeier and Pesheva 1999, Hakomori and Igarashi 1995, Lloyd and Furukawa 1998, Dulabon et al. 2000, Boldt et al. 1977, Tiemeyer et al. 1989) it is possible to suggest at least three mechanisms to explain the role of gangliosides (Fig. 1).
According to the mechanism illustrated in Fig. 1-I it is likely that specific gangliosides could interact with broadly distributed protein-based adhesion systems and favored subsets of moving cells or process that express these molecules.It is also possible to postulate a mechanism based on homophilic interaction between gangliosides located on adjacent membranes (Fig. 1-II).In addition, it has recently The ganglioside 9-O-AcG modulates the integrin receptor through a Ca ++ dependent mechanism and/or by a direct interaction between the cerebroside moiety of the ganglioside and the integrin transmembrane helix (TMH).In a first stage (a), the integrin receptor is inactive and there is no recognition of extracellular matrix (ECM) proteins.Later on (b), the gangliosides in the adjacent membrane become laterally packed around the integrin receptor (represented on the scheme by only one ganglioside), providing an optimal Ca ++ microenvironment for the activation and recognition of ECM proteins by this receptor.In addition, the gangliosides can interact directly with the integrin receptor through a conserved lysine located inside a 23 amino acids sequence at the carboxy-terminal of the TMH (outline).II: Homophilic interaction between gangliosides located at different cell membranes.In (a), two cells containing 9-O-AcG randomly spread over the membranes.In (b), the cells are interacting in a homophilic manner through a carbohydrate-carbohydrate interaction of the gangliosides.III: Heterophilic interaction between the gangliosides and a potential specific receptor.In (a), a cell containing the ganglioside 9-O-AcG in the membrane and another cell expressing a presumptive receptor.In (b), the cells are interacting by the way of a specific recognition between the ganglioside 9-O-AcG and its particular receptor.This interaction could trigger a specific cascade of reactions in both cells (not illustrated in the scheme).
been shown that selectins and/or galectins can function as receptors for specific gangliosides and this interaction could trigger a cascade of reactions in both cells (Fig. 1-III).

Fig. 1 -
Fig.1-Schematic diagrams suggesting three possible mechanisms for 9-O-acetyl GD3 (9-O-AcG) action in gliophilic or neuronophilic neuronal migration.I: The ganglioside 9-O-AcG modulates the integrin receptor through a Ca ++ dependent mechanism and/or by a direct interaction between the cerebroside moiety of the ganglioside and the integrin transmembrane helix (TMH).In a first stage (a), the integrin receptor is inactive and there is no recognition of extracellular matrix (ECM) proteins.Later on (b), the gangliosides in the adjacent membrane become laterally packed around the integrin receptor (represented on the scheme by only one ganglioside), providing an optimal Ca ++ microenvironment for the activation and recognition of ECM proteins by this receptor.In addition, the gangliosides can interact directly with the integrin receptor through a conserved lysine located inside a 23 amino acids sequence at the carboxy-terminal of the TMH (outline).II: Homophilic interaction between gangliosides located at different cell membranes.In (a), two cells containing 9-O-AcG randomly spread over the membranes.In (b), the cells are interacting in a homophilic manner through a carbohydrate-carbohydrate interaction of the gangliosides.III: Heterophilic interaction between the gangliosides and a potential specific receptor.In (a), a cell containing the ganglioside 9-O-AcG in the membrane and another cell expressing a presumptive receptor.In (b), the cells are interacting by the way of a specific recognition between the ganglioside 9-O-AcG and its particular receptor.This interaction could trigger a specific cascade of reactions in both cells (not illustrated in the scheme).