Paclitaxel binds and activates C5aR1: A new potential therapeutic target for the prevention of chemotherapy-induced peripheral neuropathy and hypersensitivity reactions

Chemotherapy-induced peripheral neuropathy (CIPN) and hypersensitivity reactions (HSRs) are among the most frequent and impairing side effects of the antineoplastic agent paclitaxel. Here, we demonstrated that paclitaxel can bind and activate complement component 5a receptor 1 (C5aR1) and that this binding is crucial in the etiology of paclitaxel-induced CIPN and anaphylaxis. Starting from our previous data demonstrating the role of interleukin (IL)-8 in paclitaxel-induced neuronal toxicity, we searched for proteins that activate IL-8 expression and, by using the Exscalate platform for molecular docking simulations, we predicted the high affinity of C5aR1 with paclitaxel. By in vitro studies, we confirmed the specific and competitive nature of the C5aR1-paclitaxel binding and found that it triggers intracellularly the NFkB/P38 pathway and c-Fos. In F11 neuronal cells and rat dorsal root ganglia, C5aR1 inhibition protected from paclitaxel-induced neuropathological effects, while in paclitaxel-treated mice, the absence (knock-out mice) or the inhibition of C5aR1 significantly ameliorated CIPN symptoms—in terms of cold and mechanical allodynia—and reduced the chronic pathological state in the paw. Finally, we found that C5aR1 inhibition can counteract paclitaxel-induced anaphylactic cytokine release in macrophages in vitro, as well as the onset of HSRs in mice. Altogether these data identified C5aR1 as a key mediator and a new potential pharmacological target for the prevention and treatment of CIPN and HSRs induced by paclitaxel.Subject terms: Pathogenesis, Immunopathogenesis     

MicroRNA miR-124 regulates neurite outgrowth during neuronal differentiation

MicroRNAs (miRNAs) are small RNAs with diverse regulatory roles. The miR-124 miRNA is expressed in neurons in the developing and adult nervous system. Here we show that overexpression of miR-124 in differentiating mouse P19 cells promotes neurite outgrowth, while blocking miR-124 function delays neurite outgrowth and decreases acetylated α-tubulin. Altered neurite outgrowth also was observed in mouse primary cortical neurons when miR-124 expression was increased, or when miR-124 function was blocked. In uncommitted P19 cells, miR-124 expression led to disruption of actin filaments and stabilization of microtubules. Expression of miR-124 also decreased Cdc42 protein and affected the subcellular localization of Rac1, suggesting that miR-124 may act in part via alterations to members of the Rho GTPase family. Furthermore, constitutively active Cdc42 or Rac1 attenuated neurite outgrowth promoted by miR-124. To obtain a broader perspective, we identified mRNAs downregulated by miR-124 in P19 cells using microarrays. mRNAs for proteins involved in cytoskeletal regulation were enriched among mRNAs downregulated by miR-124. A miR-124 variant with an additional 5′ base failed to promote neurite outgrowth and downregulated substantially different mRNAs. These results indicate that miR-124 contributes to the control of neurite outgrowth during neuronal differentiation, possibly by regulation of the cytoskeleton.     

Geldanamycin derivatives and neuroprotective effect on cultured P19-derived neurons

Geldanamycin (1), an antifungal and anticancer ansamycin, was reported as a neurotrophic and neuroprotective substance against antineoplastic drugs, paclitaxel, vincristine, and cisplatin, on cultured dorsal root ganglion neurons from chick embryos. In this study, 1 in a large quantity, together with a known 17-O-demethylgeldanamycin (2), and a new 17-O-demethylgeldanamycin hydroquinone (3) were obtained from a mangrove Streptomyces sp. A series of O-alkyl and N-alkyl derivatives of 1 were prepared by modification of C-17 and/or C-19 on the quinone ring and were evaluated for in vitro activity against P19-derived neurons. Compound 1 and 19-O-methylgeldanamycin (7) at a very low dose (1nM) enhanced survival and neurite outgrowth of P19-derived neurons and prevented neurotoxicity of paclitaxel and vinblastine. Compound 7, possessing the lowest cytotoxicity and neurotoxicity, is serving as the most promising candidate in neurodegenerative therapy against neurotoxic anticancer drugs.     

Neurite outgrowth in response to transfected N-CAM and N-cadherin reveals fundamental differences in neuronal responsiveness to CAMs

Different neuronal populations were used to compare the neurite outgrowth-promoting activities of N-CAM and N-cadherin expressed via gene transfer on the surface of nonneuronal cells. In contrast to a previously reported developmental loss of retinal ganglion cell responsiveness to N-CAM, these cells exhibited an increased and maintained responsiveness to N-cadherin over the same developmental period (E6-E11). N-CAM and N-cadherin responses could be specifically inhibited by their own antibodies, but not by antisera to the beta 1 integrin family or the L1/G4 glycoprotein. Cerebellar neurons showed qualitative differences in the nature of the dose-response curves for transfected N-CAM expression (highly cooperative) versus N-cadherin expression (linear). In addition "subthreshold" levels of N-CAM expression, which do not normally support neurite outgrowth, did so when coexpressed with functional levels of N-cadherin. These studies show fundamental differences in neuronal responsiveness to cell adhesion molecules and suggest a more dynamic regulation for N-CAM-dependent neurite outgrowth than for N-cadherin-dependent outgrowth.     

Rapid Differentiation of Human Embryonal Carcinoma Stem Cells (NT2) into Neurons for Neurite Outgrowth Analysis

Human neurons derived from stem cells can be employed as in vitro models to predict the potential of neurochemicals affecting neurodevelopmental cellular processes including proliferation, migration, and differentiation. Here, we developed a model of differentiating human neurons from well characterized human embryonal carcinoma stem cells (NT2). NT2 cells were induced to differentiate into neuronal phenotypes after 2 weeks of treatment with retinoic acid in aggregate culture. Nestin positive progenitor cells migrate out of NT2 aggregates and differentiate into βIII-tubulin expressing neuronal cells. Culturing the NT2 cells for an additional 7–14 days resulted in increased percentage of βIII-tubulin expressing cells, elaborating a long neurite that positively stained for axonal marker (Tau) and presynaptic protein (synapsin). We then asked whether neurite outgrowth from NT2 cells is modulated by bioactive chemicals. Since the cAMP/PKA pathway has been widely investigated as a regulator of neurite outgrowth/regeneration in several experimental systems, we used chemical activators and inhibitors of cAMP/PKA pathway in the culture. The adenylyl cyclase activator, forskolin, and cell-permeable analog of cAMP, 8-Br-cAMP increased the percentage of neurite bearing cells and neurite extension. Application of the protein kinase A inhibitors, H-89 and Rp-cAMP, blocked neurite formation. Taken together, NT2 aggregates undergo migration, differentiation, and neurite elaboration and can be used as a model of differentiating human neurons to screen neurochemicals and to understand cellular mechanisms of human nerve cell development.     

Identification of a neurite outgrowth-promoting domain of laminin using synthetic peptides

We have identified a synthetic peptide derived from the B2-chain of mouse laminin, Arg-Asn-Ile-Ala-Glu-Ile-Ile-Lys-Asp-Ile (p20), which stimulates the neurite outgrowth-promoting activity of the native molecule. In organotypic cultures, neurons from newborn mouse brain or embryonic peripheral nervous system responded by extensive neurite outgrowth for native laminin or the peptide p20 in the culture medium. If rat cerebellar neurons were grown on laminin, 1-5 microM (1-5 micrograms/ml) of peptide p20 in the culture medium competed with laminin and inhibited neuronal attachment and neurite outgrowth, whereas higher concentrations (greater than 50 microM; greater than 50 micrograms/ml) had a specific neurotoxic effect. When peptide p20 was used as the culture substratum, neurite outgrowth in cerebellar cultures was up to 60% of that seen on native laminin. Our results indicate that a neurite outgrowth-promoting domain of laminin is located in the alpha-helical region of the B2-chain, and is active for both central and peripheral neurons.     

Sarm1 activation produces cADPR to increase intra-axonal Ca++ and promote axon degeneration in PIPN

Cancer patients frequently develop chemotherapy-induced peripheral neuropathy (CIPN), a painful and long-lasting disorder with profound somatosensory deficits. There are no effective therapies to prevent or treat this disorder. Pathologically, CIPN is characterized by a “dying-back” axonopathy that begins at intra-epidermal nerve terminals of sensory neurons and progresses in a retrograde fashion. Calcium dysregulation constitutes a critical event in CIPN, but it is not known how chemotherapies such as paclitaxel alter intra-axonal calcium and cause degeneration. Here, we demonstrate that paclitaxel triggers Sarm1-dependent cADPR production in distal axons, promoting intra-axonal calcium flux from both intracellular and extracellular calcium stores. Genetic or pharmacologic antagonists of cADPR signaling prevent paclitaxel-induced axon degeneration and allodynia symptoms, without mitigating the anti-neoplastic efficacy of paclitaxel. Our data demonstrate that cADPR is a calcium-modulating factor that promotes paclitaxel-induced axon degeneration and suggest that targeting cADPR signaling provides a potential therapeutic approach for treating paclitaxel-induced peripheral neuropathy (PIPN).     

Distinct molecular interactions mediate neuronal process outgrowth on non-neuronal cell surfaces and extracellular matrices

We have compared neurite outgrowth on extracellular matrix (ECM) constituents to outgrowth on glial and muscle cell surfaces. Embryonic chick ciliary ganglion (CG) neurons regenerate neurites rapidly on surfaces coated with laminin (LN), fibronectin (FN), conditioned media (CM) from several non-neuronal cell types that secrete LN, and on intact extracellular matrices. Neurite outgrowth on all of these substrates is blocked by two monoclonal antibodies, CSAT and JG22, that prevent the adhesion of many cells, including neurons, to the ECM constituents LN, FN, and collagen. Neurite outgrowth is inhibited even on mixed LN/poly-D-lysine substrates where neuronal attachment is independent of LN. Therefore, neuronal process outgrowth on extracellular matrices requires the function of neuronal cell surface molecules recognized by these antibodies. The surfaces of cultured astrocytes, Schwann cells, and skeletal myotubes also promote rapid process outgrowth from CG neurons. Neurite outgrowth on these surfaces, though, is not prevented by CSAT or JG22 antibodies. In addition, antibodies to a LN/proteoglycan complex that block neurite outgrowth on several LN-containing CM factors and on an ECM extract failed to inhibit cell surface-stimulated neurite outgrowth. After extraction with a nonionic detergent, Schwann cells and myotubes continue to support rapid neurite outgrowth. However, the activity associated with the detergent insoluble residue is blocked by CSAT and JG22 antibodies. Detergent extraction of astrocytes, in contrast, removes all neurite- promoting activity. These results provide evidence for at least two types of neuronal interactions with cells that promote neurite outgrowth. One involves adhesive proteins present in the ECM and ECM receptors on neurons. The second is mediated through detergent- extractable macromolecules present on non-neuronal cell surfaces and different, uncharacterized receptor(s) on neurons. Schwann cells and skeletal myotubes appear to promote neurite outgrowth by both mechanisms.     

The heparan sulfate proteoglycan agrin modulates neurite outgrowth mediated by FGF‐2

Although the role of agrin in the formation of the neuromuscular junction is well established, other functions for agrin have remained elusive. The present study was undertaken to assess the role of agrin in neurite outgrowth mediated by the heparin‐binding growth factor basic fibroblast growth factor (FGF‐2), which we have shown previously to bind to agrin with high affinity and that has been shown to mediate neurite outgrowth from a number of neuronal cell types. Using both an established neuronal cell line, PC12 cells, and primary chick retina neuronal cultures, we find that agrin potentiates the ability of FGF‐2 to stimulate neurite outgrowth. In PC12 cells and retinal neurons agrin increases the efficacy of FGF‐2 stimulation of neurite outgrowth mediated by the FGF receptor, as an inhibitor of the FGF receptor abolished neurite outgrowth in the presence of agrin and FGF‐2. We also examined possible mechanisms by which agrin may modulate neurite outgrowth, analyzing ERK phosphorylation and c‐fos phosphorylation. These studies indicate that agrin augments a transient early phosphorylation of ERK in the presence of FGF‐2, and augments and sustains FGF‐2 mediated increases in c‐fos phosphorylation. These data are consistent with established mechanisms where heparan sulfate proteoglycans such as agrin may increase the affinity between FGF‐2 and the FGF receptor. In summary, our studies suggest that neural agrin contributes to the establishment of axon pathways by modulating the function of neurite promoting molecules such as FGF‐2. © 2003 Wiley Periodicals, Inc. J Neurobiol 55: 261–277, 2003     

GTP Hydrolysis of TC10 Promotes Neurite Outgrowth through Exocytic Fusion of Rab11- and L1-Containing Vesicles by Releasing Exocyst Component Exo70

The use of exocytosis for membrane expansion at nerve growth cones is critical for neurite outgrowth. TC10 is a Rho family GTPase that is essential for specific types of vesicular trafficking to the plasma membrane. Recent studies have shown that TC10 and its effector Exo70, a component of the exocyst tethering complex, contribute to neurite outgrowth. However, the molecular mechanisms of the neuritogenesis-promoting functions of TC10 remain to be established. Here, we propose that GTP hydrolysis of vesicular TC10 near the plasma membrane promotes neurite outgrowth by accelerating vesicle fusion by releasing Exo70. Using Förster resonance energy transfer (FRET)-based biosensors, we show that TC10 activity at the plasma membrane decreased at extending growth cones in hippocampal neurons and nerve growth factor (NGF)-treated PC12 cells. In neuronal cells, TC10 activity at vesicles was higher than its activity at the plasma membrane, and TC10-positive vesicles were found to fuse to the plasma membrane in NGF-treated PC12 cells. Therefore, activity of TC10 at vesicles is presumed to be inactivated near the plasma membrane during neuronal exocytosis. Our model is supported by functional evidence that constitutively active TC10 could not rescue decrease in NGF-induced neurite outgrowth induced by TC10 depletion. Furthermore, TC10 knockdown experiments and colocalization analyses confirmed the involvement of Exo70 in TC10-mediated trafficking in neuronal cells. TC10 frequently resided on vesicles containing Rab11, which is a key regulator of recycling pathways and implicated in neurite outgrowth. In growth cones, most of the vesicles containing the cell adhesion molecule L1 had TC10. Exocytosis of Rab11- and L1-positive vesicles may play a central role in TC10-mediated neurite outgrowth. The combination of this study and our previous work on the role of TC10 in EGF-induced exocytosis in HeLa cells suggests that the signaling machinery containing TC10 proposed here may be broadly used for exocytosis.     

A Time Course Analysis of the Electrophysiological Properties of Neurons Differentiated from Human Induced Pluripotent Stem Cells (iPSCs)

Many protocols have been designed to differentiate human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs) into neurons. Despite the relevance of electrophysiological properties for proper neuronal function, little is known about the evolution over time of important neuronal electrophysiological parameters in iPSC-derived neurons. Yet, understanding the development of basic electrophysiological characteristics of iPSC-derived neurons is critical for evaluating their usefulness in basic and translational research. Therefore, we analyzed the basic electrophysiological parameters of forebrain neurons differentiated from human iPSCs, from day 31 to day 55 after the initiation of neuronal differentiation. We assayed the developmental progression of various properties, including resting membrane potential, action potential, sodium and potassium channel currents, somatic calcium transients and synaptic activity. During the maturation of iPSC-derived neurons, the resting membrane potential became more negative, the expression of voltage-gated sodium channels increased, the membrane became capable of generating action potentials following adequate depolarization and, at day 48–55, 50% of the cells were capable of firing action potentials in response to a prolonged depolarizing current step, of which 30% produced multiple action potentials. The percentage of cells exhibiting miniature excitatory post-synaptic currents increased over time with a significant increase in their frequency and amplitude. These changes were associated with an increase of Ca²⁺ transient frequency. Co-culturing iPSC-derived neurons with mouse glial cells enhanced the development of electrophysiological parameters as compared to pure iPSC-derived neuronal cultures. This study demonstrates the importance of properly evaluating the electrophysiological status of the newly generated neurons when using stem cell technology, as electrophysiological properties of iPSC-derived neurons mature over time.     

Role of Cdc42 in neurite outgrowth of PC12 cells and cerebellar granule neurons

Inactivation of Rho GTPases inhibited the neurite outgrowth of PC12 cells. The role of Cdc42 in neurite outgrowth was then studied by selective inhibition of Cdc42 signals. Overexpression of ACK42, Cdc42 binding domain of ACK-1, inhibited NGF-induced neurite outgrowth in PC12 cells. ACK42 also inhibited the neurite outgrowth of PC12 cells induced by constitutively activated mutant of Cdc42, but not Rac. These results suggest that Cdc42 plays an important role in mediating NGF-induced neurite outgrowth of PC12 cells. Inhibition of neurite outgrowth was also demonstrated using a cell permeable chimeric protein, penetratin-ACK42. A dominant negative mutant of Rac, RacN17 inhibited Cdc42-induced neurite outgrowth of PC12 cells suggesting that Rac acts downstream of Cdc42. Further studies, using primary-cultures of rat cerebellar granule neurons, showed that Cdc42 is also involved in the neurite outgrowth of cerebellar granule neurons. Both penetratin-ACK42 and Clostridium difficile toxin B, which inactivates all members of Rho GTPases strongly inhibited the neurite outgrowth of cerebellar granule neurons. These results show that Cdc42 plays a similar and essential role in the development of neurite outgrowth of PC12 cells and cerebellar granule neurons. These results provide evidence that Cdc42 produces signals that are essential for the neurite outgrowth of PC12 cells and cerebellar granule neurons. These authors contributed equally     

The heparan sulfate proteoglycan agrin modulates neurite outgrowth mediated by FGF-2

Although the role of agrin in the formation of the neuromuscular junction is well established, other functions for agrin have remained elusive. The present study was undertaken to assess the role of agrin in neurite outgrowth mediated by the heparin-binding growth factor basic fibroblast growth factor (FGF-2), which we have shown previously to bind to agrin with high affinity and that has been shown to mediate neurite outgrowth from a number of neuronal cell types. Using both an established neuronal cell line, PC12 cells, and primary chick retina neuronal cultures, we find that agrin potentiates the ability of FGF-2 to stimulate neurite outgrowth. In PC12 cells and retinal neurons agrin increases the efficacy of FGF-2 stimulation of neurite outgrowth mediated by the FGF receptor, as an inhibitor of the FGF receptor abolished neurite outgrowth in the presence of agrin and FGF-2. We also examined possible mechanisms by which agrin may modulate neurite outgrowth, analyzing ERK phosphorylation and c-fos phosphorylation. These studies indicate that agrin augments a transient early phosphorylation of ERK in the presence of FGF-2, and augments and sustains FGF-2 mediated increases in c-fos phosphorylation. These data are consistent with established mechanisms where heparan sulfate proteoglycans such as agrin may increase the affinity between FGF-2 and the FGF receptor. In summary, our studies suggest that neural agrin contributes to the establishment of axon pathways by modulating the function of neurite promoting molecules such as FGF-2.     

Explant cultures of teleost spinal cord: source of neurite outgrowth

The source of neurite outgrowth in explant cultures of normal adult Apteronotus spinal cord was examined. Explants which contained the central region of spinal cord, including ependyma, showed neurite outgrowth in culture. Explants which did not contain ependyma showed no neurite outgrowth. It is concluded that the ependymal region is necessary for neurite outgrowth in these cultures of adult teleost spinal cord. In addition, our failure to observe axon outgrowth clearly attributable to fluorescently back-labeled electromotor neurons in these cultures suggests that the exuberant neurite outgrowth in vitro is most probably due to cells other than the electromotor neurons. This explant culture system provides a unique opportunity to study neuronal differentiation, regeneration, and neurogenesis in vitro.     

Nitric oxide-cGMP signaling regulates axonal elongation during optic nerve regeneration in the goldfish in vitro and in vivo

Nitric oxide (NO) signaling results in both neurotoxic and neuroprotective effects in CNS and PNS neurons, respectively, after nerve lesioning. We investigated the role of NO signaling on optic nerve regeneration in the goldfish ( Carassius auratus ). NADPH diaphorase staining revealed that nitric oxide synthase (NOS) activity was up-regulated primarily in the retinal ganglion cells (RGCs) 5–40 days after axotomy. Levels of neuronal NOS (nNOS) mRNA and protein also increased in the RGCs alone during this period. This period (5–40 days) overlapped with the process of axonal elongation during regeneration of the goldfish optic nerve. Therefore, we evaluated the effect of NO signaling molecules upon neurite outgrowth from adult goldfish axotomized RGCs in culture. NO donors and dibutyryl cGMP increased neurite outgrowth dose-dependently. In contrast, a nNOS inhibitor and small interfering RNA, specific for the nNOS gene, suppressed neurite outgrowth from the injured RGCs. Intra-ocular dibutyryl cGMP promoted the axonal regeneration from injured RGCs in vivo . None of these molecules had an effect on cell death/survival in this culture system. This is the first report showing that NO-cGMP signaling pathway through nNOS activation is involved in neuroregeneration in fish CNS neurons after nerve lesioning.     

Calcium influx into neurons can solely account for cell contact- dependent neurite outgrowth stimulated by transfected L1

We have used monolayers of control 3T3 cells and 3T3 cells expressing transfected human L1 as a culture substrate for rat PC12 cells and rat cerebellar neurons. PC12 cells and cerebellar neurons extended longer neurites on human L1 expressing cells. Neurons isolated from the cerebellum at postnatal day 9 responded equally as well as those isolated at postnatal day 1-4, and this contrasts with the failure of these older neurons to respond to the transfected human neural cell adhesion molecule (NCAM). Human L1-dependent neurite outgrowth could be blocked by antibodies that bound to rat L1 and, additionally, the response could be fully inhibited by pertussis toxin and substantially inhibited by antagonists of L- and N-type calcium channels. Calcium influx into neurons induced by K+ depolarization fully mimics the L1 response. Furthermore, we show that L1- and K+(-)dependent neurite outgrowth can be specifically inhibited by a reduction in extracellular calcium to 0.25 microM, and by pretreatment of cerebellar neurons with the intracellular calcium chelator BAPTA/AM. In contrast, the response was not inhibited by heparin or by removal of polysialic acid from neuronal NCAM both of which substantially inhibit NCAM-dependent neurite outgrowth. These data demonstrate that whereas NCAM and L1 promote neurite outgrowth via activation of a common CAM-specific second messenger pathway in neurons, neuronal responsiveness to NCAM and L1 is not coordinately regulated via posttranslational processing of NCAM. The fact that NCAM- and L1-dependent neurite outgrowth, but not adhesion, are calcium dependent provides further evidence that adhesion per se does not directly contribute to neurite outgrowth.     

Roles of 5-HT on phase transition of neurite outgrowth in the identified serotoninergic neuron C1, <Emphasis Type="Italic">Helisoma trivolvis</Emphasis>

Neurite outgrowth is a morphological marker of neuronal differentiation and neuroregeneration, and the process includes four essential phases, namely initiation, elongation, guidance and cessation. Intrinsic and extrinsic signaling molecules seem to involve morphological changes of neurite outgrowth via various cellular signaling cascades phase transition. Although mechanisms associated with neurite outgrowth have been studied extensively, little is known about how phase transition is regulated during neurite outgrowth. 5-HT has long been studied with regard to its relationship to neurite outgrowth in invertebrate and vertebrate culture systems, and many studies have suggested 5-HT inhibits neurite elongation and growth cone motility, in particular, at the growing parts of neurite such as growth cones and filopodia. However, the underlying mechanisms need to be investigated. In this study, we investigated roles of 5-HT on neurite outgrowth using single serotonergic neurons C1 isolated from Helisoma trivolvis. We observed that 5-HT delayed phase transitions from initiation to elongation of neurite outgrowth. This study for the first time demonstrated that 5-HT has a critical role in phase-controlling mechanisms of neurite outgrowth in neuronal cell cultures.     

Computer vision profiling of neurite outgrowth dynamics reveals spatiotemporal modularity of Rho GTPase signaling

Rho guanosine triphosphatases (GTPases) control the cytoskeletal dynamics that power neurite outgrowth. This process consists of dynamic neurite initiation, elongation, retraction, and branching cycles that are likely to be regulated by specific spatiotemporal signaling networks, which cannot be resolved with static, steady-state assays. We present NeuriteTracker, a computer-vision approach to automatically segment and track neuronal morphodynamics in time-lapse datasets. Feature extraction then quantifies dynamic neurite outgrowth phenotypes. We identify a set of stereotypic neurite outgrowth morphodynamic behaviors in a cultured neuronal cell system. Systematic RNA interference perturbation of a Rho GTPase interactome consisting of 219 proteins reveals a limited set of morphodynamic phenotypes. As proof of concept, we show that loss of function of two distinct RhoA-specific GTPase-activating proteins (GAPs) leads to opposite neurite outgrowth phenotypes. Imaging of RhoA activation dynamics indicates that both GAPs regulate different spatiotemporal Rho GTPase pools, with distinct functions. Our results provide a starting point to dissect spatiotemporal Rho GTPase signaling networks that regulate neurite outgrowth.