In the hippocampus, NCAM-deficient mice show normal levels of synaptic transmission and minor abnormalities in the paired-pulse facilitation, detectible only at 1.5 mM extracellular Ca2+ but not at higher Ca2+ concentrations50. However, NCAM-/- NMJs appears to be very interesting in this respect, since they lack paired-pulse facilitation and fail to maintain transmitter output with repetitive stimuli70. Experiments using electrical stimulation-induced loading of synaptic terminal terminals with the styryl fluorescent dye FM1-43 revealed that terminals can be loaded and destained more quickly in NCAM-/- mice than in wild-type controls71. Furthermore, clusters of dye-loaded vesicles were observed not only at the end plate but also at the preterminal part of axon, as was previously found in immature axons. As in the case of quantal release from immature axons in wild-type animals, which has been shown to be blocked by brefeldin A, preterminal loading of nerves in mature NCAM-/- junctions could also be blocked by this compound. Furthermore, application of brefeldin A strongly reduced cyclical periods of total transmission failures in NCAM-/- mice. This phenotype could be mimicked in NCAM+/+ mice by inhibitors of myosin light chain kinase (MLCK).
Examination of mice deficient in the NCAM180 revealed that the immature vesicle cycling-transmitter release system was downregulated at NMJs in the absence of NCAM 180 as in wild-type mice, but vesicle cycling was not confined to presynaptic active zones apposed to muscle72. NCAM 180 isoform-deficient synapses were also unable to sustain transmitter output with repetitive stimuli like NCAM-/- mice, despite normal Ca2+ influx into presynaptic terminals. Similar to mice lacking all NCAM isoforms, the expression of several presynaptic molecules was reduced. Among them were the calcium channel subunit Cav2.1, syntaxin, and Rab 3-interacting molecule (RIM), a molecule involved in the modulation of synaptic transmission.
Recent evidence supports the pharmacological data on involvement of MLCK in NCAM-mediated mechanisms. A highly conserved C-terminal (KENESKA) domain on NCAM was found to be required to maintain effective transmission via a pathway involving MLCK and probably myosin light chain (MLC) and myosin II73. By introducing peptides into adult NMJs, the hypothesized role of proteins in this pathway was tested by competitive disruption of protein-protein interactions. The effects of the KENESKA and other peptides on MLCK and MLC activation and on transmission failures in both wild-type and NCAM180-deficient junctions supported the role for NCAM in this pathway. Furthermore, serine phosphorylation of the KENESKA peptide appeared to be critical for this NCAM function. Thus, this pathway is required to replenish synaptic vesicles during high levels of exocytosis by facilitating myosin-driven delivery of synaptic vesicles to active zones for subsequent exocytosis. Recent data also demonstrate that NCAM-/- mice exhibit deficits in catecholamine granule trafficking between the readily releasable pool and the highly release-competent immediately releasable pool74. In summary, NCAM appears to play a fundamental role in the transmitter release mechanism found in neuroendocrine cells, at the NMJ and, possibly in central synapse active zones.
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