A DEFICIENCY OF THE SMALL GTPASE RAB8 INHIBITS MEMBRANE TRAFFIC IN DEVELOPING NEURONS

One of the major activities of developing neurons is the transport of new membrane to the growing axon. Candidates for playing a key role in the regulation of this intense traffic are the small GTP-binding prot eins of the rab family. We have used hippocampal neurons in culture an d analyzed membrane traffic activity after suppressing the expression of the small GTP-binding protein rab8. Inhibition of protein expressio n was accomplished by using sequence-specific antisense oligonucleotid es. While rab8 depletion resulted in the blockage of morphological mat uration in 95% of the neurons, suppression of expression of another ra b protein, rab3a, had no effect, and all neurons developed normal axon s and dendrites. The impairment of neuronal maturation by rab8 antisen se treatment was due to inhibition of membrane traffic. Thus, by using video-enhanced differential interference contrast microscopy, we obse rved in the rab8-depleted cells a dramatic reduction in the number of vesicles undergoing anterograde transport. Moreover, by incubating ant isense-treated neurons with Bodipy-labeled ceramide, a fluorescent mar ker for newly formed exocytic vesicles, we observed fluorescence label ing restricted to the Golgi apparatus, whereas in control cells labeli ng was found also in the neurites. These results show the role of the small GTPase rab8 in membrane traffic during neuronal process outgrowt h.