Rac1 and Cdc42 but Not RhoA or Rho Kinase Activities Are Required for Neurite Outgrowth Induced by the Netrin-1 Receptor DCC (Deleted in Colorectal Cancer) in N1E-115 Neuroblastoma Cells

Abstract

Netrins are chemotropic guidance cues that attract or repel growing axons during development. DCC (deleted in colorectal cancer), a transmembrane protein that is a receptor for netrin-1, is implicated in mediating both responses. However, the mechanism by which this is achieved remains unclear. Here we report that Rho GTPases are required for embryonic spinal commissural axon outgrowth induced by netrin-1. Using N1E-115 neuroblastoma cells, we found that both Rac1 and Cdc42 activities are required for DCC-induced neurite outgrowth. In contrast, down-regulation of RhoA and its effector Rho kinase stimulates the ability of DCC to induce neurite outgrowth. In Swiss 3T3 fibroblasts, DCC was found to trigger actin reorganization through activation of Rac1 but not Cdc42 or RhoA. We detected that stimulation of DCC receptors with netrin-1 resulted in a 4-fold increase in Rac1 activation. These results implicate the small GTPases Rac1, Cdc42, and RhoA as essential components that participate in signaling the response of axons to netrin-1 during neural development.Netrins are a small family of secreted proteins that guide growing axons during neural development (1, 2). The first netrin cloned, UNC-6, was identified using a genetic screen for mutations affecting axon guidance in Caenorhabditis elegans (3). Netrins were first identified in vertebrates on the basis of their ability to promote commissural axon outgrowth from explants of embryonic spinal cord (4, 5). Netrin family members have now been identified in multiple vertebrate and invertebrate species and shown to have a highly conserved function as axon guidance cues (6). Netrins are bifunctional molecules attracting and repelling different classes of axons. Growth cone attraction mediated by netrin-1 involves the transmembrane netrin receptor DCC (7,8). In C. elegans, the identification of UNC-5 first implicated it as a receptor required for the repellent response to UNC-6 (9). Three UNC-5 homologs have now been identified in mammals (10, 11). Current evidence suggests that netrin-mediated repulsion requires the function of both UNC-5 and DCC family members in some and perhaps all cases, suggesting that UNC-5 and DCC may form a netrin receptor complex (12).The intracellular mechanisms mediating the response of an axon to netrin-1 are currently unclear. Previous studies indicate that extracellular guidance cues induce the neuronal growth cone to advance, retract, or turn by regulating the actin cytoskeleton within the growth cone (13). The Rho family of small GTPases, in particular, RhoA, Rac1, and Cdc42, are well established regulators of actin reorganization in non-neuronal cells (14), and there is now compelling evidence demonstrating roles for RhoA, Rac1, and Cdc42 as signaling elements within the neuronal growth cone (15, 16). Here, we report that members of the Rho family of GTPases are required for commissural axon outgrowth produced by netrin-1 from explants of embryonic rat spinal cord. Both Rac1 and Cdc42 are required for neurite outgrowth promoted by DCC ...

Regulation of netrin-1 receptors by amphetamine in the adult brain

“…It has been demonstrated that stimulant drugs enhance the ratio of AMPA/NMDA receptor-mediated glutamate neurotransmission in the VTA (Borgland et al, 2004;Boudreau and Wolf, 2005;Faleiro et al, 2004;Saal et al, 2003;Sarti et al, 2007;Ungless et al, 2001), an effect that depends on NMDA receptor neurotransmission at the time of drug treatment (Ungless et al, 2001). It has recently been shown that activity of the Rac1 Rho GTPase, a downstream netrin-1 effector (Li et al, 2002), enhances excitatory synaptic transmission by promoting AMPA receptor clustering (Wiens et al, 2005). Thus, the upregulation of DCC and UNC-5 receptors in the VTA may be involved in AMPH-induced plasticity of VTA DA circuitry by enhancing the availability of AMPA receptors at the synapse.…”

“…shown that activity of the Rac1 Rho GTPase, a downstream netrin-1 effector (Li et al, 2002), enhances excitatory synaptic transmission by promoting AMPA receptor clustering (Wiens et al, 2005). Thus, the upregulation of DCC and UNC-5 receptors in the VTA may be involved in AMPH-induced plasticity of VTA DA circuitry by enhancing the availability of AMPA receptors at the synapse.…”

“…Based on these findings we propose that if netrin-1 signaling participates in the development of sensitization to AMPH, it may act in concert with bFGF to promote reorganization of VTA DA circuitry. Both bFGF and netrin-1 can induce reorganization of the actin cytoskeleton via receptor-mediated communication with the Rac1 member of the Rho family of small GTPases, key regulators of the actin cytoskeleton (Li et al, 2002;Shekarabi et al, 2005;Shin et al, 2002Shin et al, , 2004. Furthermore, it has been demonstrated that netrin-1 can induce reorganization of dendritic structure in mature neurons via Rac1 signaling (Nakayama et al, 2000).…”

Netrin requires focal adhesion kinase and Src family kinases for axon outgrowth and attraction

“…A screen for genes controlling motor axon guidance led to the isolation of max-1, which genetically interacts with unc-5 and unc-6 but not with unc-40 in C. elegans 30 . In vertebrates, netrin signaling may involve phospholipase C (PLC) 31 , phosphoinositol-3-kinase (PI3K) 31 , MAP kinases 31-33 and the small GTPases Cdc42 and Rac 34,35 . FAK is a cytoplasmic protein tyrosine kinase that was discovered a decade ago [36][37][38][39][40] . FAK is involved in multiple cellular processes including adhesion, spreading, migration, survival, cell cycle progression and proliferation 41 .…”

A dual promoter lentiviral vector for the in vivo evaluation of gene therapeutic approaches to axon regeneration after spinal cord injury

“…Netrin-1 binding to DCC activates Cdc42 and Rac1 in spinal commissural neurons 54,55 and dominant-negative Rac1 and Cdc42 inhibit DCC-mediated neurite outgrowth in neuroblastoma cells, whereas inhibition of RhoA or its effector Rho kinase increases axon extension in response to netrin-1. 56 Predominance of RhoA activation resulting from the expression of myelin inhibitory proteins and their receptors in the adult CNS 57-59 may overwhelm effects of myrDCCDP1 expression. Thus, although overexpression of myrDCCDP1 from our dual promoter lentiviral vector in rubrospinal and corticospinal axons in the adult spinal cord was not sufficient to promote axon regeneration, these experiments did show the feasibility of our approach in quantifying axonal growth responses to therapeutic gene candidate expression.…”