Role of cell adhesion molecule DM-GRASP in growth and orientation of retinal ganglion cell axons

Abstract

The cell adhesion molecule (CAM) DM-GRASP was investigated with respect to a role for axonal growth and navigation in the developing visual system. Expression analysis reveals that DM-GRASP's presence is highly spatiotemporally regulated in the chick embryo retina. It is restricted to the optic fiber layer (OFL) and shows an expression maximum in a phase when the highest number of retinal ganglion cell (RGC) axons extend. In the developing retina, axons grow between the DM-GRASP-displaying OFL and the Laminin-rich basal lamina. We show that DM-GRASP enhances RGC axon extension and growth cone size on Laminin substrate in vitro. Preference assays reveal that DM-GRASP-containing lanes guide RGC axons, partially depending on NgCAM in the axonal membrane. Inhibition of DM-GRASP in organ-cultured eyes perturbs orientation of RGC axons at the optic fissure. Instead of leaving the retina, RGC axons cross the optic fissure and grow onto the opposite side of the retina. RGC axon extension per se and navigation from the peripheral retina towards the optic fissure, however, is not affected. Our results demonstrate a role of DM-GRASP for axonal pathfinding in an early phase of the formation of the higher vertebrate central nervous system.

Ubiquitination and Endocytosis of Cell Adhesion Molecule DM-GRASP Regulate Its Cell Surface Presence and Affect Its Role for Axon Navigation

“…The interactions of DM-GRASP present on the substrate axons with DM-GRASP on the advancing growth cones are crucial for proper axonal orientation in the retina (9). The data presented here now show that the endocytosis of DM-GRASP is crucial for the preferential growth of RGC axons on DM-GRASP-containing micro-lanes that mimic the earlier formed RGC axons.…”

“…Retinal Single Cell Cultures-Single cell cultures of embryonic day 6 retina were prepared as described (9,35). The cultures were fixed in 4% paraformaldehyde for 30 min and stained by indirect immunofluorescence as described (36).…”

“…The proteins were separated by SDS-polyacrylamide gel electrophoresis and then transferred to a nitrocellulose sheet. Western blot analysis with a polyclonal DM-GRASP antibody was performed as described before (9).…”

“…trans-interaction, we employed a substrate preference assay (Fig. 4) offering alternating lanes of DM-GRASP plus laminin versus laminin only as axonal growth substrate (9,35). This assay reflects the situation of RGC axons navigating in the developing retina where they selectively extend in the optic fiber layer, a structure composed of RGC axons and the only layer providing DM-GRASP; laminin is ubiquitously present.…”

“…The IgSF-CAM DM-GRASP (2) (also termed SC1 (3), BEN (4), and JC7 (5)) has been shown to play a role in cell adhesion (2,6), axon growth (7,8), axon navigation (9,10) as well as in neuronal migration (11), differentiation (12), and synapse formation (13,14). Highly conserved mammalian orthologs of DM-GRASP have been found in human (ALCAM/CD166 (15)), rodents (ALCAM/CD166 (16,17)), and bovine (CD166 (18)).…”