Rho inhibition recruits DCC to the neuronal plasma membrane and enhances axon chemoattraction to netrin 1

Abstract

Molecular cues, such as netrin 1, guide axons by influencing growth cone motility. Rho GTPases are a family of intracellular proteins that regulate the cytoskeleton, substrate adhesion and vesicle trafficking. Activation of the RhoA subfamily of Rho GTPases is essential for chemorepellent axon guidance; however, their role during axonal chemoattraction is unclear. Here, we show that netrin 1, through its receptor DCC, inhibits RhoA in embryonic spinal commissural neurons. To determine whether netrin 1-mediated chemoattraction requires Rho function, we inhibited Rho signaling and assayed axon outgrowth and turning towards netrin 1. Additionally, we examined two important mechanisms that influence the guidance of axons to netrin 1: substrate adhesion and transport of the netrin receptor DCC to the plasma membrane. We found that inhibiting Rho signaling increased plasma membrane DCC and adhesion to substrate-bound netrin 1, and also enhanced netrin 1-mediated axon outgrowth and chemoattractive axon turning. Conversely, overexpression of RhoA or constitutively active RhoA inhibited axonal responses to netrin 1. These findings provide evidence that Rho signaling reduces axonal chemoattraction to netrin 1 by limiting the amount of plasma membrane DCC at the growth cone, and suggest that netrin 1-mediated inhibition of RhoA activates a positive-feedback mechanism that facilitates chemoattraction to netrin 1. Notably, these findings also have relevance for CNS regeneration research. Inhibiting RhoA promotes axon regeneration by disrupting inhibitory responses to myelin and the glial scar. By contrast, we demonstrate that axon chemoattraction to netrin 1 is not only maintained but enhanced, suggesting that this might facilitate directing regenerating axons to appropriate targets.

A Signaling Mechanism Coupling Netrin-1/Deleted in Colorectal Cancer Chemoattraction to SNARE-Mediated Exocytosis in Axonal Growth Cones

“…It has been shown that Netrin-1 triggers membrane exposure of DCC-containing vesicles in growth cones (Bouchard et al, 2004;Moore et al, 2008). First, we corroborated that incubation with Netrin-1, but not with control media or BDNF (which acts via TrkB receptors), caused the mobilization of DCC from the inside of growth cones to the cell membrane ( Fig.…”

“…For instance, chemoattractant guidance cues may lead to increased exocytosis, thereby resulting in membrane extension in a given direction of growth. Recent studies have shown that PKA (protein kinase A) activation recruits DCC to the plasma membrane and enhances axon chemoattraction to Netrin-1 (Bouchard et al, 2004;Moore et al, 2008). Indeed, recent studies showed that local increases of Ca 2ϩ result in asymmetric exocytosis in the growth cone (Tojima et al, 2007) and that clathrin-mediated endocytosis is essential for Semaphorin 3A-induced chemorepulsion (Tojima et al, 2010).…”

Neuron Subpopulations with Different Elongation Rates and DCC Dynamics Exhibit Distinct Responses to Isolated Netrin-1 Treatment

“…4A and 4B); consistent with earlier work using cortical neurons. [36][37][52][53] The presence of receptors in all subcellular regions is in agreement with the notion that neurons may be sensing differences in Netrin-1 concentration along their entire lengths to modulate their elongation rates, and across their growth cones to modulate turning responses. Signal intensities were measured at three axonal regions of interest (ROIs): axon shafts, growth cones and filopodia (Fig.…”

c-Jun N-terminal Kinase 1 (JNK1) Is Required for Coordination of Netrin Signaling in Axon Guidance

“…Netrin-1 stimulation recruits multiple signal transduction molecules, such as Fyn, FAK, PAK1, P130 CAS , TRIO, and DOCK180, through DCC to form a signaling complex (41, 43, 68 -70), which then activates small GTPases Rac1 and Cdc42 (44,46,63,64,(71)(72)(73) or inhibits RhoA in Netrin-induced attraction (74). Toxin B, an inhibitor of the small GTPases Rho, Rac, and Cdc42, not only blocked Netrin-1-induced Rac1 activity but inhibited the induction of phospho-JNK1 by Netrin-1 without affecting phospho-p38 (Fig.…”