Functional role of the neural cel adhesion molecule NCAM and novel interaction partners

Abstract

In this thesis, I show that the intracellular domain (ICD) of the neural cell adhesion molecule (NCAM) and the bacterial homolog of polysialic acid (PSA), colominic acid (CA), interact with the N-terminal domain of the transient receptor potential canonical (TRPC) channels 1, -4, and -5. The NCAM-PSA-TRPC interaction is shown by immunoprecipitation, pull-down, and ELISA experiments, confirming that NCAM140, NCAM180, and CA interact with TRPC1, -4, and -5. Moreover, the interaction between NCAM-PSA and TRPC1, -4, and -5 is further verified by co-localization experiments and proximity ligation assay (PLA) using cultured cerebellar, hippocampal, and cortical neurons. By total internal reflection fluorescence microscopy, I show that those interactions occur at the plane or near the plasma membrane but not in the endoplasmic reticulum. Furthermore, the interaction of NCAM with TRPC1, -4, and -5 occurs independently of PSA on NCAM because the treatment of cells with Endo N, which degrades PSA, does not affect the localization of the interaction. In NCAM-expressing CHO 2A10 cells or NCAM-PSA-expressing CHO C6 cells, the treatment with an NCAM-triggering antibody (NCAM-Ab) only increases the Ca2+ flux in CHO C6 cells overexpressing TRPC1/4 or TRPC1/5. Moreover, the application of a general TRPC inhibitor, SKF-96365, reduces the NCAM-dependent Ca2+ flux after overexpression of TRPC1/4 or TRPC1/5, suggesting that the Ca2+ flux induced by NCAM-Ab treatment in NCAM-PSA-expressing CHO cells is modulated by TRPC1, -4, and -5. The calcium imaging experiments in cortical cells show that the NCAM-dependent Ca2+ flux is only triggered in cells expressing NCAM-PSA. NCAM-deficient cells and wild-type cells treated with Endo N show a reduction in the Ca2+ flux. Furthermore, pre-treatment of wild-type cells with the TRPC1, -4, and -5 inhibitor, pico145, and CA reduce the NCAM-dependent Ca2+ flux. The pre-treatment of cells with thapsigargin, an inhibitor of the retrotranslocation of calcium to the intracellular stores, in the absence or presence of Ca2+ in the medium, reveals that the NCAM-dependent signaling is mainly triggered by extracellular calcium. Finally, the NCAM-dependent Erk1/2 phosphorylation and the NCAM-dependent neurite outgrowth are also reduced in cells pre-treated with TRPC1, -4, and -5 inhibitors. Altogether, these results suggest a role of TRPC1, -4, and -5 in the regulation of NCAM-dependent neurite outgrowth.