NGL-3-induced presynaptic differentiation of hippocampal neurons in an afadin-dependent, nectin-1-independent manner.

A hippocampal mossy fiber synapse, which is implicated in learning and memory, has a complex structure. We have previously shown using afadin-deficient mice that afadin plays multiple roles in the structural and functional differentiations of this synapse. We investigated here using a co-culture system with cultured hippocampal neurons and non-neuronal COS-7 cells expressing synaptogenic cell adhesion molecules (CAMs) whether afadin is involved in the presynaptic differentiation of hippocampal synapses. Postsynaptic CAMs NGL-3 (alias, a Lrrc4b gene product) and neuroligin induced presynaptic differentiation by trans-interacting with their respective presynaptic binding CAMs LAR (alias, a Ptprf gene product) and neurexin. This activity of NGL-3, but not neuroligin, was dependent on afadin, but not the afadin-binding presynaptic CAM nectin-1. The afadin-binding postsynaptic CAM nectin-3 did not induce presynaptic differentiation. Immunofluorescence and immunoelectron microscopy analyses showed that afadin was localized mainly at puncta adherentia junctions, but partly at synaptic junctions, of the mossy fiber synapse. ß-Catenin and γ-catenin known to bind to LAR were co-immunoprecipitated with afadin from the lysate of mouse brain. These results suggest that afadin is involved in the NGL-3-LAR system-induced presynaptic differentiation of hippocampal neurons cooperatively with ß-catenin and γ-catenin in a nectin-1-independent manner.