Clustering of the Neural Cell Adhesion Molecule (NCAM) at the Neuronal Cell Surface Induces Caspase-8- and -3-dependent Changes of the Spectrin Meshwork Required for NCAM-mediated Neurite Outgrowth

Doreen Westphal,Vladimir Sytnyk,Melitta Schachner,Iryna Leshchyns'Ka

doi:10.1074/jbc.m110.177147

Abstract

Changes in neuronal morphology underlying neuronal differentiation depend on rapid and sustained cytoskeleton rearrangements in the growing neurites. Whereas cell adhesion molecules are well established as regulators of neuronal differentiation, less is known about the signaling mechanisms by which they influence the cytoskeleton. Here we show that the neural cell adhesion molecule (NCAM) associates with the active form of caspase-8 and that clustering of NCAM at the neuronal cell surface leads to activation of caspase-8 and -3 followed by the cleavage of the sub-membranous brain spectrin meshwork, but not of the actin or tubulin cytoskeleton. Inhibitors of caspase-8 and -3 specifically block the NCAM-dependent spectrin cleavage and abolish NCAM-dependent neurite outgrowth. NCAM-dependent rearrangements of the membrane associated spectrin meshwork via caspase-8 dependent caspase-3 activation are thus indispensable for NCAM-mediated neurite outgrowth.

Highlights

Acts with multiple binding partners on adjacent cells and in the extracellular matrix, including adhesion molecules, such as prion protein (PrP), L1, and neural cell adhesion molecule (NCAM) itself [5, 6], growth factor receptors, such as FGFR and GFR␣ [7, 8], and other receptors, such as receptor-type protein phosphatase ␤ or its secreted forms [9]
Profiles of protein band densities calculated within the blot area marked with a curly bracket are shown on the right and were used to quantify differences
Graph shows quantitation of blots from several experiments with the levels of cleaved ␤II spectrin normalized to the total levels of ␤II spectrin. *, p Ͻ 0.05, paired t test

Summary

Introduction

Acts with multiple binding partners on adjacent cells and in the extracellular matrix, including adhesion molecules, such as prion protein (PrP), L1, and NCAM itself [5, 6], growth factor receptors, such as FGFR and GFR␣ [7, 8], and other receptors, such as receptor-type protein phosphatase ␤ or its secreted forms [9]. To analyze whether the association of NCAM with caspase-8 depends on NCAM clustering at the cell surface, we used indirect immunofluorescence labeling to compare distributions of NCAM and caspase-8 in cultured hippocampal mouse neurons treated with NCAM antibodies to cluster NCAM at the cell surface or with nonspecific immunoglobulins as control.

Results

Conclusion