Functional role of a polysialic acid-carrying proteolytic fragment of the neural cell adhesion molecule NCAM in the nervous system

The membrane-associated glycoprotein NCAM is the main carrier of the unusual glycan α2,8-linked polysialic acid (PSA) in the mammalian nervous system. PSA-NCAM is involved in the regulation of differentiation and migration of neuronal precursor cells, axonal outgrowth, synaptogenesis, physiological and morphological synaptic plasticity in the early embryonic stage during brain development. In the adulthood, PSA-NCAM is upregulated after injury of the central and peripheral nervous system and enhances axon regrowth in the peripheral nervous system and sprouting in the central nervous system. Moreover, PSA-NCAM plays an important role in the control of the circadian rhythm. PSA-NCAM can participate in homophilic and/or heterophilic interactions which can mediate signal transduction pathways. These NCAM-mediated signal transduction pathways can be modified by PSA due to its biophysical characteristics. Several proteins have been reported to interact with the extracellular or intracellular domain of NCAM while only a few binding partners are known to interact with PSA. In the present study the generation and nuclear import of a transmembrane PSA-carrying NCAM fragment has been shown upon treatment of cerebellar neurons with surrogate NCAM ligands, e.g. function-triggering NCAM antibody or NCAM-Fc, with the PSA ligand FGF-2 or with a peptide comprising the effector domain (ED) of MARCKS. The results of immunoblot analysis and cell surface biotinylation have revealed that this PSA-carrying NCAM fragment is proteolytically cleaved from full length NCAM at the plasma membrane by the matrix metalloproteases MMP2 and/or MMP9. Treatment of cerebellar neurons with surrogate NCAM and/or PSA ligands leads to activation of a FGF-receptor-mediated PLC-dependent and PKC-dependent signal transduction pathway as well as phosphorylation of MARCKS. Interestingly, the generation and nuclear import of the PSA-carrying NCAM fragment by NCAM antibody and FGF-2 was shown to depend on calmodulin-mediated activation of NOS, which enhanced production and release of NO and led to NO-dependent S-nitrosylation of MMP9 and activation of MMP2, while the MARCKS-ED peptide triggered generation and nuclear import of the PSA-carrying NCAM fragment depends on the activation of PLD- and PI3K-mediated signaling pathways which activate MMP2. To unravel the pathway by which the PSA-NCAM fragment reaches the nucleus, immunoelectron microscopy and subcellular fractionation of stimulated cells after cell surface biotinylation were performed. The results demonstrated that the PSA-carrying NCAM fragment is transported to endosomes after internalization. Moreover, this fragment is translocated from the endosomes into the cytoplasm in a calmodulin-dependent manner and the import into the nucleus was shown to depend on cofilin and PC4, but not calmodulin. In this study, cofilin and PC4 were identified as novel binding partners of PSA by performing immunoprecipitation with PSA antibody and ELISA. Moreover, the nuclear co-localization of PSA with cofilin and PC4 and histone H1 was confirmed by proximity ligation assay. The nuclear PSA-NCAM levels varied in different brain regions depending on the 12 h light/ 12 h dark cycle. Nuclear PSA was shown to be involved in the regulation of mRNA levels of the clock-related genes circadian locomotor output cycles kaput (CLOCK) and period-1 (Per-1) during the circadian rhythm. Moreover, microarray analysis revealed an influence of the nuclear PSA-carrying as well as PSA-lacking NCAM fragment on the regulation of mRNA levels of nuclear receptor subfamily 2 group F member 6 (Nr2f6) or of low density lipoprotein receptor-related protein 2 (Lrp2) and α-synuclein. The upregulation of Nr2f6 was found to depend on nuclear PSA-NCAM levels, while the upregulation of Lrp2 and α-synuclein depended on nuclear NCAM levels. Together, these findings demonstrate the relevance of the nuclear PSA-NCAM fragment in transcription and affirmed that nuclear PSA-NCAM plays an important role in the circadian rhythm, indicating a functional relevance of nuclear PSA-NCAM in the nervous system.