Directed cell migration and axonal guidance are essential steps in neural development. Both processes are controlled by specific guidance cues that activate the signaling cascades that ultimately control cytoskeletal dynamics. Another essential step in migration and axonal guidance is the regulation of plasmalemma turnover and exocytosis in leading edges and growth cones. However, the cross talk mechanisms linking guidance receptors and membrane exocytosis are not understood. Netrin-1 is a chemoattractive cue required for the formation of commissural pathways. Here, we show that the Netrin-1 receptor deleted in colorectal cancer (DCC) forms a protein complex with the t-SNARE (target SNARE) protein Syntaxin-1 (Sytx1). This interaction is Netrin-1 dependent both in vitro and in vivo, and requires specific Sytx1 and DCC domains. Blockade of Sytx1 function by using botulinum toxins abolished Netrin-1-dependent chemoattraction of axons in mouse neuronal cultures. Similar loss-offunction experiments in the chicken spinal cord in vivo using dominant-negative Sytx1 constructs or RNAi led to defects in commissural axon pathfinding reminiscent to those described in Netrin-1 and DCC loss-of-function models. We also show that Netrin-1 elicits exocytosis at growth cones in a Sytx1-dependent manner. Moreover, we demonstrate that the Sytx1/DCC complex associates with the v-SNARE (vesicle SNARE) tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) and that knockdown of TI-VAMP in the commissural pathway in the spinal cord results in aberrant axonal guidance phenotypes. Our data provide evidence of a new signaling mechanism that couples chemotropic Netrin-1/DCC axonal guidance and Sytx1/TI-VAMP SNARE proteins regulating membrane turnover and exocytosis.

Total lack of visual experience [dark rearing (DR)] is known to prolong the critical period and delay development of sensory functions in mammalian visual cortex. Recent results show that neurotrophins (NTs) counteract the effects of DR on functional properties of visual cortical cells and exert a strong control on critical period duration. NTs are known to modulate the development and synaptic efficacy of neurotransmitter systems that are affected by DR. However, it is still unknown whether the actions of NTs in dark-reared animals involve interaction with neurotransmitter systems. We have studied the effects of DR on the expression of key molecules in the glutamatergic and GABAergic systems in control and NT-treated animals. We have found that DR reduced the expression of the NMDA receptor 2A subunit and its associated protein PSD-95 (postsynaptic density-95), of GRIP (AMPA glutamate receptor interacting protein), and of the biosynthetic enzyme GAD (glutamic acid decarboxylase). Returning dark-reared animals to light for 2 hr restored normal expression of the above-mentioned proteins almost completely. NT treatment specifically counteracts DR effects; NGF acts primarily on the NMDA system, whereas BDNF acts primarily on the GABAergic system. Finally, the action of NT4 seems to involve both excitatory and inhibitory systems. These data demonstrate that different NTs counteract DR effects by modulating the expression of key molecules of the excitatory and inhibitory neurotransmitter systems.

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Directed cell migration and axonal guidance are essential steps in neural development that share many molecular mechanisms. The guidance of developing axons and migrating neurons is likely to depend on the precise control of plasmalemma turnover in selected regions of leading edges and growth cones, respectively. Previous results provided evidence of a signaling mechanism that couples chemotropic deleted in colorectal cancer (DCC)/Netrin-1 axonal guidance and exocytosis through Syntaxin1(Sytx1)/TI-VAMP SNARE proteins. Here we studied whether Netrin-1-dependent neuronal migration relies on a similar SNARE mechanism. We show that migrating neurons in the lower rhombic lip (LRL) express several SNARE proteins, and that DCC co-associates with Sytx1 and TI-VAMP in these cells. We also demonstrate that cleavage of Sytx1 by botulinum toxin C1 (BoNT/C1) abolishes Netrin-1-dependent chemoattraction of migrating neurons, and that interference of Sytx1 functions with shRNAs or Sytx1-dominant negatives disrupts Netrin-1-dependent chemoattraction of LRL neurons. These findings indicate that a Sytx1/DCC interaction is required for Netrin-1 guidance of migrating neurons, thereby highlighting a relationship between guidance signaling and SNARE proteins that regulate membrane turnover.