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.

Glioblastoma (GBM) is the most prevalent adult brain tumor, with virtually no cure, and with a median overall survival of 15 months from diagnosis despite of the treatment. SNARE proteins mediate membrane fusion events in cells and are essential for many cellular processes including exocytosis and neurotransmission, intracellular trafficking and cell migration. Here we show that the blockade of the SNARE protein Syntaxin 1 (Stx1) function impairs GBM cell proliferation. We show that Stx1 loss-of-function in GBM cells, through ShRNA lentiviral transduction, a Stx1 dominant negative and botulinum toxins, dramatically reduces the growth of GBM after grafting U373 cells into the brain of immune compromised mice. Interestingly, Stx1 role on GBM progression may not be restricted just to cell proliferation since the blockade of Stx1 also reduces in vitro GBM cell invasiveness suggesting a role in several processes relevant for tumor progression. Altogether, our findings indicate that the blockade of SNARE proteins may represent a novel therapeutic tool against GBM.

Total lack of visual experience (dark rearing, DR) is known to affect development of mammalian visual cortex (VC) and to prolong the critical period of visual cortical plasticity. Neurotrophins (NTs) have been proposed to play a relevant role in activity dependent processes important for the final shaping of cortical visual connections. Neurotrophin supply or antagonism of endogenous NT action profoundly affect visual cortical development and plasticity; in particular, exogenous supply of NTs counteracts DR effects on VC development. However, the effects of DR on NT expression are still debated and mounting evidence reports a mismatch between BDNF mRNA and protein expression in DR animals. To gain insight into the effects of DR on expression of nerve growth factor (NGF) and the functional state of NT signalling pathways, we assessed the phosphorylation state of Trk receptors in light-reared animals (LR), in dark-reared animals (DR), in DR animals briefly exposed to light and in DR animals with exogenous supply of NTs [NGF, brain-derived neurotrophic factor (BDNF) and NT-4] in the VC. We report that DR increases the expression of NGF but reduces the phosphorylation of TrkA and TrkB receptors with respect to LR; normal phosphorylation is rapidly rescued by a brief exposure to light. Exogenous supply of NGF, BDNF or NT4 in DR animals also rescues the phosphorylation of their receptors.

Axonal guidance and synaptic specification depends on specific signaling mechanisms that occur in growth cones. While several signaling pathways implicated in cone navigation have been identified, membrane dynamics in growth cones remains largely unknown. We took advantage of SynaptopHluorin and high-speed optical recordings to monitor the patterns of membrane dynamics in rat hippocampal growth cones. We show that exocytosis occurs both at the peripheral and central domains, including filopodia, and that SynaptopHluorin signals occur as spontaneous patterned peaks. Such transients average approximately two per minute and last ϳ30 s. We also demonstrate that the chemoattractant Netrin-1 elicits increases in the frequency and slopes of these transients, with peaks averaging up to six per minute in the peripheral domain. Netrin-1-dependent regulation of exocytotic events requires the activation of the Erk1/2 and SFK pathways. Furthermore, we show that domains with high SynaptopHluorin signals correlate with high local calcium concentrations and that local, spontaneous calcium increases are associated with higher SynaptopHluorin signals. These findings demonstrate highly stereotyped, spontaneous transients of local exocytosis in growth cones and that these transients are positively regulated by chemoattractant molecules such as Netrin-1.

Neurotrophins are involved in many crucial cellular functions, including neurite outgrowth, synapse formation, and plasticity. Although these events have long been known, the molecular determinants underlying neuritogenesis have not been fully characterized. Ack1 (activated Cdc42-associated tyrosine kinase) is a non-receptor tyrosine kinase that is highly expressed in the brain. Here, we demonstrate that Ack1 is a molecular constituent of neurotrophin signaling cascades in neurons and PC12 cells. We report that Ack1 interacts with Trk receptors and becomes tyrosine phosphorylated and its kinase activity is increased in response to neurotrophins. Moreover, our data indicate that Ack1 acts upstream of the Akt and MAPK pathways. We show that Ack1 overexpression induces neuritic outgrowth and promotes branching in neurotrophin-treated neuronal cells, whereas the expression of Ack1 dominant negatives or short-hairpin RNAs counteract neurotrophin-stimulated differentiation. Our results identify Ack1 as a novel regulator of neurotrophin-mediated events in primary neurons and in PC12 cells.