R80 Dispatch

Axon guidance: GTPases help axons reach their targetsGianluca Gallo and Paul C. Letourneau

During development and regeneration of the nervoussystem, axons must correctly navigate to their specifictargets through a complex molecular environment.Recent work has shed light on how GTPases of the Rasfamily are involved in transducing extracellular signalsinto responses that lead to directed neurite outgrowth.

Address: Department of Cell Biology and Neuroanatomy, 4-144Jackson Hall, The University of Minnesota, Minneapolis, Minnesota55455, USA.

Current Biology 1998, 8:R80–R82http://biomednet.com/elecref/09609822008R0080

© Current Biology Ltd ISSN 0960-9822

Axons and dendrites, collectively referred to as neurites,navigate through the complex environment of the devel-oping embryo using a highly specialized structure each ofthem has at its end, termed the growth cone. Growthcones detect and respond to information from their imme-diate environment. In order to sample molecules in theirvicinity, growth cones extend filopodia and lamellae thatare endowed with sensitive receptors. Although we havelearned much about the cytoskeletal mechanisms thatdrive motile activity and about the extracellular signalsthat regulate the dynamics of cell behavior, relatively little

is known about how growth cones transduce an extracellu-lar signal into behavioral responses (Figure 1) [1].

One class of molecules whose study promises to yield sub-stantial insights into axon guidance is the family of Ras-related GTPases, including Rho, Rac and Cdc42. TheseGTPases have been shown to be involved in the reorgani-zation of the actin cytoskeleton and in the generation ofcell extensions — filopodia and lamellae — in non-neu-ronal cells (reviewed in [2]), suggesting that these proteinsmight also have a role in neuronal growth cones. In thisarticle, we highlight some recent findings regarding thefunction of GTPases in the regulation of growth cone mor-phology and neurite outgrowth. We will first discuss thefindings of recent studies in vitro, using both neuronal celllines and primary neurons, and then data obtained fromstudies of GTPase function in vivo, during axonal growth.

Tales from the culture dishA direct approach has suggested that tensile forcesexerted by growth cones have an important role in regu-lating the coordinated assembly and reorganization of theneurite cytoskeleton. When a calibrated micropipette tipcoated with laminin is touched to the surface of a neuron,it adheres; as the micropipette is slowly pulled away, a

Figure 1

Microtubules

[Ca2+]

Receptor (tyrosine phosphorylation ?)

Receptor

MAPs

MAPs

Tubulin

[Ca2+]Myosins

Rac, Rho, Cdc42 G actin

ABPs

F actin

CAM/SAM

?

??

?

Growth factor or other ligand

GAP 43

[Ca2+]

Current Biology

ActinKinases

Phosphatases

IP3, PIP

GAP

43

The growth cone components involved in navigation. Navigationinvolves three steps: detection of guidance cues, cytoplasmicsignaling, and motile responses. Each growth cone componentpictured here can be assigned to one of these steps, as follows.Detection involves perhaps a growth factor or other ligand and theappropriate receptor, as well as cell adhesion molecules/substratumadhesion molecules (CAMs/SAMs). Cytoplasmic signaling may involve

Ca2+, protein kinases and phosphatases, Rac, Rho, Cdc42, inositoltrisphosphate (IP3), phosphatidyl inositol phosphate (PIP), andreceptor tyrosine kinases and phosphatases. Motile responses involvemicrotubules, actin, myosins, microtubule-associated proteins (MAPs)and actin-binding proteins (ABPs), as well as the membrane-associated growth cone protein GAP43.

neurite is drawn out from the cell. In a similar manner,the tensions generated by growth cone motility induceneurite formation. Lamoureux et al. [3] have investigatedthe role of Rac in such tension-mediated neurite out-growth by expressing a dominant-negative mutant of Racin PC12 phaeochromocytoma cells that had been primedwith nerve growth factor (NGF). The expression of domi-nant-negative mutant Rac inhibited normal, growth-cone-mediated neurite growth induced by NGF, but theformation of tension-induced neurites from PC12 cellsdid not differ between cells transfected with the mutantRac and controls. Lamoureux and colleagues [3] con-cluded that, in PC12 cells, Rac is involved in some aspectof growth cone attachment to the substratum or in theproduction of growth cone tension, but not in the conse-quent reorganization of the microtubule cytoskeleton andextension of the neurite.

How do the three best-characterized GTPases — Rho,Rac1 and Cdc42 — interact in regulating axonal growth? Arequirement for Rac1 and Cdc42 in neurite outgrowth andthe production of filopodia and lamellae in N1E-115 neu-roblastoma cells has been reported by Kozma et al. [4], whoalso investigated the role of Rho in neurite outgrowth andretraction. Neurite outgrowth was induced in neuroblas-toma cells by microinjection of Clostridium botulinum C3exoenzyme, a toxin which specifically inactivates Rho. C3-induced outgrowth was blocked by co-injection of a domi-nant-negative mutant of either Rac1 or Cdc42. Similarly,dominant-negative mutants of Rac1 and Cdc42 alsoblocked neurite outgrowth in response to serum starvation.The role of Rho as an inhibitor of neurite outgrowth isfurther supported by the findings of Kozma et al. [4] andothers, that inactivation of Rho by C3 also blocks growthcone collapse induced by the serum signaling moleculelysophosphatidic acid, whereas microinjection of Rho or aconstitutively active form of Rho causes growth cone col-lapse. Kozma et al. [4] sum up their findings by suggestingthat Rho is an inhibitor of neurite outgrowth whereasCdc42 acts upstream of Rac1 and promotes neurite out-growth by regulating the formation of growth cone filopo-dia and lamellae. Thus, neurite outgrowth may beregulated as the Rho and Cdc42/Rac pathways withingrowth cones compete, perhaps for a limited resource,resulting in either the establishment of growth cone lamel-lar and filopodial structures (via the Cdc42/Rac pathway)or the loss of these structures (via the Rho pathway).

The studies described above used model neuronal celllines, and it is not always clear that such cell lines faithfullyreproduce the activities of primary neurons. Although thedata regarding the roles of GTPases in neurite outgrowthin PC12 and N1E-115 cells are in agreement, a study usingembryonic chick dorsal root ganglion (DRG) neurons pro-duced different results. Jin and Strittmatter [5] report thatC3 stimulates axonal outgrowth from DRG neurons, but

the growth cones of DRG neurons under these conditionsare devoid of lamellae and filopodia, in contrast to the C3-induced growth cone formation in N1E-115 cells reportedby Kozma et al. [4]. Furthermore, in C3-treated DRGneurons, the introduction of constitutively active Rhoincreased growth cone spreading and decreased the rate ofneurite outgrowth. On the other hand, constitutivelyactive Rac1 in DRG cells increased the proportion of col-lapsed growth cones, in contrast to the Rac1-mediatedlamellar formation observed with N1E-115 cells.

Jin and Strittmatter [5] also investigated the role ofGTPases in the growth cone collapse mediated by therepellent guidance cue collapsin, and found that col-lapsin’s actions on growth cones are mediated via Rac1and not Rho or Cdc42. Given that neither the growth conecollapse induced by lysophosphatidic acid nor thatinduced by myelin was prevented by dominant-negativemutant Rac1, not all the guidance cues that collapse DRGgrowth cones act through Rac1. Jin and Strittmatter [5]presented a model for the roles of Rho and Rac1 in DRGgrowth cone behavior and neurite outgrowth that involvesthree states. When outgrowth is slow and growth cones aremostly collapsed, both Rho and Rac1 are active. Whenoutgrowth is moderate and growth cones exhibit lamellaeand filopodia, Rho is active and Rac1 is inactive. Whengrowth is rapid and growth cones are mostly collapsed,Rho is inactive and Rac1 may be in either state.

Threadgill et al. [6] investigated the effects of expressingconstitutively active and dominant-negative forms ofRho, Rac and Cdc42 on dendritic development in cul-tured cortical neurons. Dendritic formation was reducedby inhibiting Rho GTPases, and the number of dendriteswas increased by expressing the constitutively activeforms of all three GTPases. Thus, the regional and mor-phological diversity of cortical neurons may arise from thedifferential activation of Rho-related GTPases. Further-more, although GTPases regulate aspects of growth conemorphology and axon growth in both model neuronalcells and primary neurons, they may do so differently, andcare must be taken in extrapolating from results with neu-ronal cell lines and between neuronal types (see Table 1).

Tales from the embryo Although studies in vitro have provided insights into theways in which GTPases are involved in neurite growth,they do not address whether GTPases are actually impor-tant in axonal and dendritic growth in vivo. Zipkin et al. [7]investigated this issue by creating mutants of the nema-tode Caenorhabditis elegans deficient in the gene encoding anew Rho family member, Mig-2. The absence of the Mig-2protein caused a variety of effects on cell migration, butonly subtle effects were noticed in the nervous system.Strikingly, the outgrowth of one identified neuron (thehermaphrodite-specific neuron, HSN) was affected by

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mutations that produced constitutively active Mig-2, butnot by Mig-2 loss-of-function mutants. In the former, theHSN extended long axons that wandered in a seeminglyhaphazard fashion along the body wall and did not followthe correct pathway. Zipkin et al. [7] interpret these effectsof the constitutively active Mig-2 as being the result ofinterference with the ability of the HSN growth cone tointerpret the normal guidance cues correctly. Althoughintriguing, the findings of Zipkin et al. [7] do not reveal anyobvious requirement for basal Mig-2 levels in vivo. Previ-ous studies of Drosophila that express constitutively activeand dominant-negative mutant GTPases (reviewed byLuo et al. [8]) found more widespread effects of Rac1 andRho on axonal and dendritic growth, however. So,GTPases have been shown to have roles in neuronal devel-opment. But given the complexity of the environment invivo and the multitude of receptor and signaling pathwaysactivated at any given time in growth cones, studies withmutants may often reveal only subtle effects, as severalredundant mechanisms could act in parallel to guide axonscorrectly, even in the absence of individual GTPases.

In summary, GTPases of the Ras superfamily have beenshown to be involved in the regulation of axonal growth.Although recent publications have provided us withinsights into the roles of Rho, Rac and Cdc42 in the mech-anisms underlying axonal growth, many questions remainunanswered. For example, by what mechanisms do extra-cellular cues activate GTPases in growing neurites? Howdo the GTPases interact with other signaling pathwaysactivated during neuritic growth, such as cell adhesionmolecules and neurotrophins? How many more GTPasesare involved in neurite growth and guidance? How do

GTPases interact — are they in series or in parallel? Howare GTPases involved in the refinement of neuronal con-nectivity patterns? Are GTPases involved only in growth-cone-mediated growth and guidance or also in theformation of collateral branches of neurons? Given theresourcefulness and creativity of investigators in this veryactive field, answers to these questions surely lie in thenot-too-distant future.

References1. Letourneau PC: The cytoskeleton in nerve growth cone motility

and axonal pathfinding. Perspect Dev Neurobiol 1996, 4:111-123.2. Tapon N, Hall A: Rho, Rac and Cdc42 GTPases regulate the

organization of the actin cytoskeleton. Curr Opin Cell Biol 1997,9:86-92.

3. Lamoureux P, Altun-Glutekin ZF, Lin C, Wagner JA, Heidemann SR:Rac is required for growth cone function but not neuriteassembly. J Cell Sci 1997, 110:635-641.

4. Kozma R, Sarner S, Ahmed S, Lim L: Rho family GTPases andneuronal growth cone remodeling: relationships betweenincreased complexity induced by Cdc42, Rac1 and acetylcholineand collapse induced by RhoA and lysophophatidic acid. Mol CellBiol 1997, 17:1201-1211.

5. Jin Z, Strittmatter SM: Rac1 mediates collapsin-1 induced growthcone collapse. J Neurosci 1997, 17:6256-6263.

6. Threadgill R, Bobb K, Ghosh A: Regulation of dendritic growth andremodeling by Rho, Rac and Cdc42. Neuron 1997, 19:625-634.

7. Zipkin ID, Kindt RM, Kenyon CJ: Role of a new Rho family memberin cell migration and axon guidance in C. elegans. Cell 1997,90:883-894.

8. Luo L, Jan LY, Jan Y-N: Rho family small GTP-binding proteins ingrowth cone signaling. Curr Opin Neurobiol 1997, 7:81-86.

Table 1

A summary of the effects of Rho, Rac and Cdc42 on neurite outgrowth in cultured cells and neurons.

GTPase Activity Cell type Effect on neurites Reference

Rho Dominant-negative (inactive) mutant Cortical neurons Decreased numbers of dendrites [6]C3 toxin injection (inactivation) NIE-115 cell line Stimulation of growth cone motility and outgrowth [4]C3 toxin injection (inactivation) DRG neurons Stimulation of outgrowth [5]

Constitutively active Cortical neurons Increased numbers of dendrites [6]Constitutively active NIE-115 cell line Growth cone collapse [4]

Rac Dominant-negative (inactive) mutant PC12 cell line Inhibition of outgrowth [3]Dominant-negative (inactive) mutant Cortical neurons Decreased numbers of dendrites [6]Dominant-negative (inactive) mutant NIE-115 cell line Inhibition of C3- or serum-induced outgrowth [4]

Constitutively active Cortical neurons Increased numbers of dendrites [6]Constitutively active NIE-115 cell line Growth cone collapse [4]Constitutively active DRG neurons Growth cone collapse [5]

Cdc42 Dominant-negative (inactive) mutant Cortical neurons Decreased numbers of dendrites [6]Dominant-negative (inactive) mutant NIE-115 cell line Inhibition of C3- or serum-induced outgrowth [4]

Constitutively active Cortical neurons Increased numbers of dendrites [6]Constitutively active NIE-115 cell line Growth cone collapse [4]