Overexpression of neuronal Sec1 enhances axonal branching in hippocampal neurons

The soluble N-ethylmaleimide-sensitive factor-attached protein receptor (SNARE) proteins syntaxin 1 and synaptosomal-associated protein-25 have been implicated in axonal outgrowth. Neuronal Sec1 (nSec1), also called murine unc18a (Munc18a), is a syntaxin 1-binding protein involved in the regulation of SNARE complex formation in synaptic vesicle membrane fusion. Here we analysed whether nSec1/Munc18a is involved in neurite formation. nSec1/Munc18a expressed under the control of an inducible promoter in differentiated PC12 cells as well as in hippocampal neurons appears first in the cell body, and at later times after induction along neurites and in growth cones. It is localised to distinct tubular and punctated structures. In addition, exogenous nSec1/Munc18a inhibited regulated secretion in PC12 cells. Overexpression in PC12 cells of nSec1/Munc18a or its homologue Munc18b, reduced the total length of neurites. This effect was enhanced with nSec1-T574A, a mutant that lacks a cyclin-dependent kinase 5 phosphorylation site and displays an increased binding to syntaxin 1. In contrast, in hippocampal neurons the total length of all primary neurites and branches was increased upon transfection of nSec1/Munc18a. Detailed morphometric analysis revealed that this was a consequence of an increased number of axonal side branches, while the average lengths in primary neurites and of side branches were not affected. From these results we suggest that nSec1/Munc18a is involved in the regulation of SNARE complex-dependent membrane fusion events implicated in the ramification of axonal processes in neurons.