Axons Navigate Noise with 190RhoGAP
Authors: Pavol Zelina, R. Jeroen Pasterkamp
Journal: Neuron (2019)

In this issue of Neuron, Bonanomi et al., 2019Bonanomi D. Valenza F. Chivatakarn O. Sternfeld M.J. Driscoll S.P. Aslanian A. Lettieri K. Gullo M. Badaloni A. Lewcock J.W. et al.P190RhoGAP filters competing signals to resolve axon guidance conflicts.Neuron. 2019; 102 (this issue): 602-620Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar investigate how navigating axons ignore irrelevant guidance signals. They show that the binding of p190RhoGAP to DCC suppresses inappropriate responses to Netrin-1, allowing motor axons to exit the embryonic spinal cord. In this issue of Neuron, Bonanomi et al., 2019Bonanomi D. Valenza F. Chivatakarn O. Sternfeld M.J. Driscoll S.P. Aslanian A. Lettieri K. Gullo M. Badaloni A. Lewcock J.W. et al.P190RhoGAP filters competing signals to resolve axon guidance conflicts.Neuron. 2019; 102 (this issue): 602-620Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar investigate how navigating axons ignore irrelevant guidance signals. They show that the binding of p190RhoGAP to DCC suppresses inappropriate responses to Netrin-1, allowing motor axons to exit the embryonic spinal cord. During the formation of functional neural circuits, axons are guided to their targets by attractive and repulsive cues in the extracellular environment. These cues instruct axons to follow specific predetermined routes or to avoid inappropriate ones. Different families of axon guidance cues have been identified. They bind specialized cell surface receptors to activate downstream signaling pathways that induce axon turning (Kolodkin and Tessier-Lavigne, 2011Kolodkin A.L. Tessier-Lavigne M. Mechanisms and molecules of neuronal wiring: a primer.Cold Spring Harb. Perspect. Biol. 2011; 3: a001727Crossref PubMed Scopus (436) Google Scholar). Considering the complexity of the developing nervous system, a remarkably limited set of guidance cues has been identified. However, axon guidance is tightly controlled, and molecular mechanisms are in place to diversify the effects of guidance cues. Axons, for example, regulate receptor availability to dictate guidance cue sensitivity and to navigate different intermediate targets (O’Donnell et al., 2009O’Donnell M. Chance R.K. Bashaw G.J. Axon growth and guidance: receptor regulation and signal transduction.Annu. Rev. Neurosci. 2009; 32: 383-412Crossref PubMed Scopus (256) Google Scholar, Bai and Pfaff, 2011Bai G. Pfaff S.L. Protease regulation: the Yin and Yang of neural development and disease.Neuron. 2011; 72: 9-21Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Adding to the complexity of the guidance process is the fact that many neuronal classes establish their axonal connections concomitantly in an exquisitely complex cellular environment consisting of stationary cell types, migrating cells, and other growing axons. Guidance cues presented by these cellular structures may provide relevant information but also signals meant for unrelated axons with different final destinations. Key issues are, therefore, not only how axons respond to relevant cues but also how they ignore irrelevant directional signals. In this issue of Neuron, Bonanomi et al., 2019Bonanomi D. Valenza F. Chivatakarn O. Sternfeld M.J. Driscoll S.P. Aslanian A. Lettieri K. Gullo M. Badaloni A. Lewcock J.W. et al.P190RhoGAP filters competing signals to resolve axon guidance conflicts.Neuron. 2019; 102 (this issue): 602-620Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar identify the intracellular protein p190RhoGAP (p190) as an inhibitor of motor axon responses to the guidance cue Netrin-1. p190 allows motor axons to ignore pial surface-derived Netrin-1 and thereby exit the embryonic spinal cord and extend toward their peripheral targets. To identify molecular regulators of spinal motor axon guidance, Bonanomi et al., 2019Bonanomi D. Valenza F. Chivatakarn O. Sternfeld M.J. Driscoll S.P. Aslanian A. Lettieri K. Gullo M. Badaloni A. Lewcock J.W. et al.P190RhoGAP filters competing signals to resolve axon guidance conflicts.Neuron. 2019; 102 (this issue): 602-620Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar performed an unbiased forward genetics screen in mice that express GFP in their motor projections. In one of the mouse mutants they identified, named Cassin, motor axons aberrantly project toward the dorsal spinal cord close to the pial surface instead of exiting at the ventral root exit points. Other motor pathfinding defects observed in Cassin mutants include defasciculation of thoracic intercostal nerves and mistargeting of hindlimb-innervating peroneal nerves (Figure 1). The Cassin mutation maps to Arhgap35, which encodes p190, a GTPase-activating protein (GAP) that negatively regulates RhoGTPases (Settleman et al., 1992Settleman J. Albright C.F. Foster L.C. Weinberg R.A. Association between GTPase activators for Rho and Ras families.Nature. 1992; 359: 153-154Crossref PubMed Scopus (252) Google Scholar). Using different approaches, Bonanomi et al., 2019Bonanomi D. Valenza F. Chivatakarn O. Sternfeld M.J. Driscoll S.P. Aslanian A. Lettieri K. Gullo M. Badaloni A. Lewcock J.W. et al.P190RhoGAP filters competing signals to resolve axon guidance conflicts.Neuron. 2019; 102 (this issue): 602-620Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar confirmed that Cassin is indeed a new loss-of-function allele of p190 and that p190 is required in motor neurons for proper targeting of their axons. Next, they asked whether the effects of p190 on motor axon guidance are dependent on its ability to inhibit Rho GTPases. To address this question, Bonanomi et al., 2019Bonanomi D. Valenza F. Chivatakarn O. Sternfeld M.J. Driscoll S.P. Aslanian A. Lettieri K. Gullo M. Badaloni A. Lewcock J.W. et al.P190RhoGAP filters competing signals to resolve axon guidance conflicts.Neuron. 2019; 102 (this issue): 602-620Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar analyzed mice harboring a p190-R1284A mutation that renders the p190GAP domain inactive. Interestingly, p190-R1284A embryos showed intact motor axon exit from the spinal cord and normal intercostal nerves, but defective peroneal nerve targeting. This argues for a non-catalytic function of p190 in mediating spinal exit and highlights an unexpected spatiotemporal requirement for p190GAP activity in motor axon pathfinding; early stages of motor axon guidance do not require p190GAP activity; however, this activity is required later during motor axon target innervation (Figure 1). p190 mediates signaling downstream different axon guidance cues (Shimizu et al., 2008Shimizu A. Mammoto A. Italiano Jr., J.E. Pravda E. Dudley A.C. Ingber D.E. Klagsbrun M. ABL2/ARG tyrosine kinase mediates SEMA3F-induced RhoA inactivation and cytoskeleton collapse in human glioma cells.J. Biol. Chem. 2008; 283: 27230-27238Crossref PubMed Scopus (83) Google Scholar, Jeong et al., 2012Jeong S. Juhaszova K. Kolodkin A.L. The control of semaphorin-1a-mediated reverse signaling by opposing pebble and RhoGAPp190 functions in Drosophila.Neuron. 2012; 76: 721-734Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). In order to establish which signaling pathways require p190 to control motor axons, Bonanomi et al., 2019Bonanomi D. Valenza F. Chivatakarn O. Sternfeld M.J. Driscoll S.P. Aslanian A. Lettieri K. Gullo M. Badaloni A. Lewcock J.W. et al.P190RhoGAP filters competing signals to resolve axon guidance conflicts.Neuron. 2019; 102 (this issue): 602-620Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar examined the p190 interactome in embryonic spinal cord and found that several components of the Netrin signaling cascade bind p190. Netrin-1 is a well-characterized guidance cue that influences motor neuron projections (Poliak et al., 2015Poliak S. Morales D. Croteau L.P. Krawchuk D. Palmesino E. Morton S. Cloutier J.F. Charron F. Dalva M.B. Ackerman S.L. et al.Synergistic integration of Netrin and ephrin axon guidance signals by spinal motor neurons.eLife. 2015; 4: e10841Crossref PubMed Scopus (52) Google Scholar) and, interestingly, demarcated the trajectory taken by ectopic intraspinal motor axons in p190 knockout (KO) embryos. The potential involvement of Netrin-1 in generating p190 KO embryonic motor axon guidance phenotypes was further supported by expression of the Netrin-1 receptor DCC on spinal motor axons exiting the spinal cord, including those derailed dorsally in p190 KO mice. Further, p190 and DCC biochemically interact and co-localize in axonal growth cones in vitro. To understand the mechanistic link between Netrin-1/DCC and p190, Bonanomi et al., 2019Bonanomi D. Valenza F. Chivatakarn O. Sternfeld M.J. Driscoll S.P. Aslanian A. Lettieri K. Gullo M. Badaloni A. Lewcock J.W. et al.P190RhoGAP filters competing signals to resolve axon guidance conflicts.Neuron. 2019; 102 (this issue): 602-620Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar exposed wild-type and p190-deficient ventral spinal cord explants to Netrin-1. Intriguingly, whereas wild-type explants were unresponsive to Netrin-1, motor axons from those taken from p190 KO mice were attracted to the Netrin-1 source. This suggests that p190 normally attenuates Netrin-1 attraction. Strong evidence for this model was further provided by crosses between p190 KO and Netrin-1 or DCC KO mice showing that ectopic intraspinal motor axon bundles were nearly absent in double KO mice (Figure 1). Together, these data suggest that p190-DCC binding renders spinal motor axons unresponsive to the attractive effects of Netrin-1 expressed at the pial surface, allowing their spinal cord exit. This is of great interest since pial surface-derived Netrin-1 guides spinal commissural axons ventrally, indicating that, in the embryonic spinal cord, Netrin-1 is both a relevant and irrelevant guidance signal. These results underscore the necessity for axons to discriminate between instructive signals and noise. Of note, intercostal and peroneal nerve defects were still present in the above-mentioned double mutants, and none of these phenotypes were observed in Netrin-1 or DCC mutants alone. Therefore, the role of p190 during intercostal and hindlimb nerve development must rely on cooperation with other signaling systems. While axon guidance is classically viewed as the cumulation of responses to instructive signals, this study by Bonanomi et al., 2019Bonanomi D. Valenza F. Chivatakarn O. Sternfeld M.J. Driscoll S.P. Aslanian A. Lettieri K. Gullo M. Badaloni A. Lewcock J.W. et al.P190RhoGAP filters competing signals to resolve axon guidance conflicts.Neuron. 2019; 102 (this issue): 602-620Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar suggests that suppression of responses to irrelevant cues is equally important. These findings raise many intriguing questions and suggest new directions of investigation. For example, it is remarkable that an intact p190GAP domain is required for peroneal nerve development, while other aspects of motor axon pathfinding rely on non-canonical GAP-independent p190 signaling. Further, given the ubiquitous expression of p190 and the general role of Rho (which is activated in p190 KO mice), it is surprising that only a very few specific axon guidance phenotypes are observed in p190 KO mice. Another outstanding question is how p190 silences motor axon responses to Netrin-1. Does p190 restrict the recruitment of molecular effectors to DCC (Figure 1)? This model gains support from the observation that p190 binds the intracellular domain of DCC between the P2 and P3 motifs. This region binds multiple signaling molecules, including Src family tyrosine kinases (SFKs) and focal adhesion kinase (FAK), implicated in Netrin-1/DCC signaling (Boyer and Gupton, 2018Boyer N.P. Gupton S.L. Revisiting Netrin-1: one who guides (axons).Front. Cell. Neurosci. 2018; 12: 221Crossref Scopus (77) Google Scholar). It is also supported by the comparison between DCC interactomes from wild-type or p190 KO mouse embryonic stem cell (ESC)-derived motor neurons. Bonanomi et al., 2019Bonanomi D. Valenza F. Chivatakarn O. Sternfeld M.J. Driscoll S.P. Aslanian A. Lettieri K. Gullo M. Badaloni A. Lewcock J.W. et al.P190RhoGAP filters competing signals to resolve axon guidance conflicts.Neuron. 2019; 102 (this issue): 602-620Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar report that in wild-type ESC neurons, DCC associates with proteins involved in transcription and translation, whereas in p190 KO ESC neurons, DCC mainly associates with signaling proteins active in axon growth and guidance. Thus, p190 may act to prevent DCC from linking to pathways that regulate axon guidance. Future work is needed to define the mechanisms that lead to p190-mediated silencing of Netrin-1 attraction. The observation that DCC associates with proteins that constitute different signaling pathways in the absence or presence of p190 raises the attractive possibility that these pathways are mutually exclusive and that p190 acts as a molecular switch to dictate DCC’s signaling output. The question why in some neurons p190 silences Netrin-1/DCC signaling (i.e., in spinal motor neurons) while in others it appears not to (e.g., in spinal commissural neurons) remains unanswered. A detailed comparison of the molecular consequences of p190 deletion in both cell types will help to resolve this apparent discrepancy. Apart from silencing Netrin-1 attraction, p190 could influence other aspects of Netrin-1 signaling. For example, Netrin-1 and DCC can engage in adhesive interactions that may be modulated by p190. Further, p190 could influence Netrin-1 through laminin signaling. Laminin can convert Netrin-1 attraction to repulsion in retinal axons and is enriched together with Netrin-1 at the pial surface, and p190 functions downstream of laminin receptors. Therefore, examining whether loss of laminin signaling unmasks Netrin-1 attraction and contributes to intraspinal motor axon diversion is an interesting direction of further research. Overall, this study by Bonanomi et al., 2019Bonanomi D. Valenza F. Chivatakarn O. Sternfeld M.J. Driscoll S.P. Aslanian A. Lettieri K. Gullo M. Badaloni A. Lewcock J.W. et al.P190RhoGAP filters competing signals to resolve axon guidance conflicts.Neuron. 2019; 102 (this issue): 602-620Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar highlights the importance of mechanisms that allow neurons to suppress responses to distracting guidance signals. Since many axons and cells navigate in a “noisy” environment composed of relevant and irrelevant guidance signals, similar discriminatory mechanisms likely operate elsewhere within and outside of the nervous system. Further, given the contribution of guidance cue deregulation to disease (Van Battum et al., 2015Van Battum E.Y. Brignani S. Pasterkamp R.J. Axon guidance proteins in neurological disorders.Lancet Neurol. 2015; 14: 532-546Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar), it is important to understand whether an inability to distinguish signal from noise in the context of developmental axon guidance contributes to brain disease. p190RhoGAP Filters Competing Signals to Resolve Axon Guidance ConflictsBonanomi et al.NeuronMarch 19, 2019In BriefAxon growth requires an enormous signaling matrix as the underlying code for building the nervous system. In a genetic screen, Bonanomi et al. identify p190RhoGAP as a critical regulatory switch that silences contextually inappropriate guidance signals for precise wiring of neuronal connections. Full-Text PDF Open Archive