Outgrowth of neurites is a dual process

Abstract

In neurons and neurosecretory (nerve) cells, neurite outgrowth requires the enlargement of the plasma membrane sustained by the exocytosis of specific vesicles. The well known, slow canonical form of outgrowth induced in pheochromocytoma PC12 cells by NGF, as well as the outgrowth taking place in neurons, involve vesicles positive for the vSNARE Ti-VAMP. Working in defective PC12 clones expressing high levels of the transcriptional repressor REST, we have identified now a new, rapid form of outgrowth, triggered by activation of a small GTPase, Rac1. This form is sustained by the exocytosis of another type of vesicles, taking place locally at the tip of neurite growth cones, the enlargeosomes (vSNARE: VAMP4). This new form, which is positively controlled by REST, requires the dynamics of microtubules, but not of microfilaments. Its signalling remains undefined because established second messengers, (Ca2+, DAG, cAMP) seem not involved. Using a high REST/enlargeosome-rich PC12 clone transfected with TrkA we have found that the NGF-induced outgrowth is not always slow, but can be fast in cells expressing high levels of the receptor involved, TrkA; that PC12 can express together the two distinct forms of outgrowth, canonical and new, activated independently from each other. Their comparative characterization in terms of changes in the cytoskeleton has now been initiated. The two forms are present also in neurons where the new one seems to predominate in the initial phases of development, the canonical one later on. Our results identify a new aspect of the REST impact in nerve cell specificity/function. The existence of two distinct forms of neurite outgrowth may cope better than a single form with the variable needs of nerve cells in the subsequent stages of their development.

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Figures and Tables

Figure 1 Membrane traffic in growth cones of early and mature neuronal cultures. The model to the left illustrates the structure of growth cones in cultured nerve cells, phase 2 of neurite outgrowth. The external zone labeled in red corresponds to the filopodia and lamellipodia filled with actin (peripheral zone). Vesicles are few in this zone and numerous in the central gray zone, in continuity with the transitional zone. The blue vesicles to the left, labeled 1, are the enlargeosomes undergoing intense exocytosis (thick arrow); the green, large ones to the right, labeled 2, are the vesicles of the bulk endocytosis.18 Notice that their arrow is smaller than that of the exocytic vesicles because a prevalence of exocytosis is necessary for neurites to grow. The small red vesicles labeled 3 are precursors of neurosecretory vesicles, not yet competent for exo/endocytosis. The model to the right illustrates a similar growth cone belonging however to an axon in phase 3/4. Two exo/endocytic cycles are illustrated. That to the left, labeled 1, concerns the TiVAMP-positive vesicles (green) that account for the growth at this stage. The arrow of exocytosis is therefore thicker of that of endocytosis. The cycle to the right, labeled 2, concerns in contrast the small synaptic vesicles (red) and their recycling endocytic vesicles. This cycle does not contribute to the axonal growth, therefore the arrows of exo end endocytosis are of similar thickness.

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