Do fragments of the neural cell adhesion molecule NCAM with or without attached polysialic acid differentially regulate transcription?
Molecular and Cellular Neurology and Neuropathology
Final Report Abstract
Glycans on proteins regulate important interactions between cells as well as between cells and the extracellular matrix. Glycoproteins are not only found at the cell surface, but also in the nucleus, raising the question of the functional relevance of the glycan moieties in the nucleus. The glycan polymer polysialic acid (PSA) and its major carrier, the neural cell adhesion molecule NCAM, play important roles in the mammalian nervous system during development and in the adulthood by regulating cell interactions and by affecting synaptic activities and regeneration after trauma. PSA modulates the functions of NCAM and influences cellular function by direct interactions with its ligands histone H1, brain-derived neurotrophic factor, fibroblast growth factor 2 (FGF-2) and myristoylated alanine-rich C kinase substrate (MARCKS). In recent studies, we have found that PSA-lacking and -carrying proteolytic NCAM fragments comprising the intracellular and transmembrane domains as well as part of the extracellular domain enter the cell nucleus. The PSA-lacking transmembrane NCAM fragment is generated by a serine protease activity at the plasma membrane upon stimulation of cultured cerebellar neurons or transfected CHO cells with surrogate NCAM ligands and it is transferred to the cell nucleus via the endoplasmic reticulum and the cytoplasm. Generation and nuclear import of this fragment depend on calmodulin and the focal adhesion kinase. In the present project, we addressed the question by which mechanisms and pathways the PSA-carrying transmembrane NCAM fragment is generated and transported to the nucleus. In addition, we investigated in this project whether the nuclear PSA-carrying and -lacking NCAM fragments are involved in the regulation of gene expression. Using cultured murine cerebellar neurons, we showed that the FGF-2- or NCAM antibody-triggered generation of the nuclear PSA-carrying NCAM fragment depends on MARCKS phosphorylation, activation of the FGF receptor, phospholipase C, protein kinase C and phosphoinositide-3-kinase and requires calmodulin-dependent activation of nitric oxide synthase, production and release of nitric oxide, nitric oxide-dependent S-nitrosylation and activation of matrix metalloprotease 9 (MMP9) and MMP9-dependent activation of matrix metalloprotease 2 (MMP2). Generation of the nuclear PSA-NCAM fragment by a synthetic peptide comprising the effector domain of MARCKS does not depend on the calmodulin-dependent activation of nitric oxide synthase and the production and release of nitric oxide, but depends on phospholipase D activation and MMP9-independent activation of MMP2. Of note, the generation of the nuclear PSA-carrying NCAM fragment does not depend on the focal adhesion kinase. Using PSA-NCAM-expressing CHO cells, we showed that the PSA-carrying NCAM fragment is translocated to endosomes after its generation at the plasma membrane, released from the endosomes into the cytoplasm and imported from the cytoplasm into the nucleus. The release from endosomes is mediated by calmodulin, while the nuclear import is not mediated by calmodulin, but by the PSA-binding proteins Positive Cofactor 4 (PC4), which plays important roles in chromatin organization and in transcription, and cofilin, which is involved in the nuclear import of actin and in regulation of transcription. Immunoelectron microscopy of cerebellar neurons supports the notion that PSA-NCAM is translocated from the plasma membrane to the nucleus via endosomes. In the nucleus, the PSA-carrying NCAM fragment interacts with histone H1 and PC4 via PSA and both the PSA-carrying and -lacking NCAM fragment interact with histone H1 via their intracellular protein domains as indicated by proximity ligation assays. Moreover, we provide evidence that the nuclear PSA-NCAM fragment is involved in the regulation of the circadian rhythm in cerebellar neurons by affecting the expression of clock-related genes of Circadian Locomotor Output Cycles Kaput and Period-1, which both are key players in transcriptional-translational feedback loops controlling the circadian rhythm. Furthermore, we found that the expression of nuclear receptor subfamily 2 group F member 6, which plays a role in the entrainment of the circadian rhythm, is regulated on the mRNA and protein level by the nuclear PSA-carrying NCAM fragment. In contrast, the nuclear PSA-lacking NCAM fragment was shown to regulate the mRNA and protein expression of α-synuclein, which is involved in presynaptic signaling and is linked to Parkinson's disease. In addition, we showed that the nuclear PSA-lacking fragment regulates the expression of mRNA coding for low density lipoprotein receptor-related protein 2 which is a multi-ligand receptor. In summary, our results indicate the generation of the PSA-carrying and PSA-lacking NCAM fragments are mediated by different pathways and mechanisms and that the nuclear NCAM fragments regulate gene expression of different genes, suggesting that the function of the nuclear PSA-carrying NCAM fragment differs from that of the PSA-lacking NCAM fragment.
Publications
- (2016). Polysialic acid enters the cell nucleus attached to a fragment of the neural cell adhesion molecule NCAM to regulate the circadian rhythm in mouse brain. Mol Cell Neurosci 74:114-127
Westphal N, Kleene R, Lutz D, Theis T, Schachner M
(See online at https://doi.org/10.1016/j.mcn.2016.05.003)
