Changes in the localization of NAP-22, a calmodulin binding membrane protein, during the development of neuronal polarity.

Abstract

NAP-22, a neuronal tissue-enriched acidic membrane protein, is a Ca(2+)-dependent calmodulin binding protein and has similar biochemical characteristics to GAP-43 (neuromodulin). Recent biochemical studies have demonstrated that NAP-22 localizes in the membrane raft domain with a cholesterol-dependent manner. Since the raft domain is assumed to be important to establish and/or to maintain the cell polarity, we have investigated the changes in the localization of NAP-22 during the development of the neuronal polarity in vitro and in vivo, using cultured hippocampal neurons and developing cerebellum neurons, respectively. Cultured hippocampal neurons initially extended several short processes, and at this stage NAP-22 was distributed more or less evenly among them. During the maturation of neuronal cells, NAP-22 was sorted preferentially into the axon. Throughout the developmental stages of hippocampal neurons, the localization change of NAP-22 was quite similar to that of tau, an axonal marker protein, but not to that of microtubule-associated protein-2 (MAP-2), a dendritic marker protein. Further confocal microscopic observation demonstrated the colocalization of NAP-22 and either tau or vesicle-associated protein-2 (VAMP-2). A comparison of the time course of the axonal localization of NAP-22 and GAP-43 showed that NAP-22 localization was much later than that of GAP-43. The correlation between the expression of NAP-22 and synaptogenesis in the cerebellar granular layer, particularly in the synaptic glomeruli, was also investigated. There existed many VAMP-2 positive synapses but no NAP-22 positive ones in 1-week-old cerebellum. On sections of 2-week-old cerebellum, accumulation of NAP-22 to the synaptic glomeruli was clearly observed and this accumulation became clearer during the maturation of the synaptic structure. The present results suggest the possibility that NAP-22 plays an important role in the maturation and/or the maintenance of synapses rather than in the process of the axonal outgrowth, by controlling cholesterol-dependent membrane dynamics.