Small GTPases of Rho family play critical roles the cytoskeleton regulation.  Many molecules that are known to regulate the cytoskeleton, such as cofilin, profiling, gelsolin and Arp2/3, are the downstream effectors of Rho GTPases.  The three main family members that are well studied are Cdc42, Rac1, and RhoA.  Cdc42 and Rac1 have similar effects on cytoskeleton.  They both promote the actin polymerization in the cell and profit the formation of filopodia and lamelipodia respectively.  RhoA’s effect may be opposite to Cdc42 and Rac, which increases the contraction of cell periphery or cell processes, and cause the cell rounding.  Through their regulation on cytoskeleton, Rho GTPases are involved in many cellular processes, including cell division, cell migration, nerve growth, cell secretion, and vesicle transport.  Many in vitro and in vivo studies have shown that Rho GTPase mutations change the neuronal morphology, the nerve growth, as well as the axon projection, suggesting a role played by Rho GTPases in axon pathfinding, while how these happen awaits further clarification.

In the present study, we used the growth cone turning assay developed by Mu-ming Poo to examine the role of Rho family GTPases in mediating axon guidance in Xenopus spinal neurons by brain-derived neurotrophic factor (BDNF), and Lysophosphatidic acid (LPA), which are secreted factors existing in the developing brain, as well as ryanodine, a drug that regulates the internal calcium store. 

BDNF is a neurotrophic factor widely expressed in the brain, which promotes nerve growth, cell survival, axon regeneration, and neuronal migration.  In vivo and in vitro studies have shown that BDNF is a candidate chemoattractant to nerve growth cones which works through its specific receptor Trk B.  Since BDNF promotes nerve growth, we guess that Cdc42 and Rac may function in its signaling processes.  Ryanodine is a naturally occurring alkaloid which regulates the calcium release from the internal calcium store.  At low concentrations, ryanodine binds to and opens the internal calcium channel, while at high concentrations, it blocks the intracellular calcium channels.  When a gradient of ryanodine is applied to the growth cone, it triggers attraction at low concentraton and repulsion at high concentration.  Lysophosphatidic acid (LPA), a bioactive lipid known to induce proliferative and/or morphological effects on neurons, has been proposed to be an extracellular signal involved in early neurogenesis.  It acts through specific G protein-coupled, seven-transmembrane domain receptors Ga12/13 or Gq, and increases intracellular RhoA activity.  In neuronal cells, activation of RhoA by LPA results in axon growth inhibition and growth cone collapse.

Using the growth cone turning assay, we found that gradients of BDNF and LPA can induce attraction and repulsion to growth cones respectively.  Low concentration  ryanodine can mimic BDNF to induce growth cone attraction and high dose ryanodine  gradient is repulsive to the growth cone.  To further study the role of Rho GTPases in growth cone turning, GTPase mutants，either dominant negative (DN) or constitutive active (CA), were expressed in Xenopus spinal neurons by injection of the cDNA of the GFP-fusion protein into one of the blastomeres of Xenopus embryos at 2- or 4-cell stage.  The effects of these mutants on the growth cone turning triggered by BDNF, LPA and ryanodine were observed to determine whether Rho GTPases are directly involved in axon guidance.  We found that, expression of either DN-Cdc42 or CA-Cdc42 in cultured Xenopus spinal neurons abolished the chemoattractive turning of their growth cones in a gradient of BDNF, and low concentration  ryanodine, whereas chemorepulsive turning induced by a gradient of LPA was abolished by the expression of DN-RhoA and converted to chemoattraction by the expression of CA-Cdc42.  Conversely, repulsive turning induced by BDNF in the presence of an inhibitor of cAMP pathway was also converted to attraction in DN-Cdc42 or DN-RhoA expressing neurons.  High dose ryanodine gradient was rather ineffective in triggering any turning response in neurons expressing DN-Cdc42, CA-Cdc42 or DN-RhoA.  These data suggest that Cdc42 can mediate attractive signal triggered by a gradient of BDNF or low concentration ryanodine, whereas RhoA can mediate repulsion of LPA, and that there is crosstalk between Cdc42 and RhoA pathways, which only influence the growth cone repulsion.  Furthermore, the uniform presence of Y-27632, the specific inhibitor of ROCK, which is the direct RhoA downstream kinase, blocked the LPA-induced repulsion, and a gradient of Y-27632 by itself is capable of inducing attractive turning，suggesting that asymmetric RhoA or ROCK activity in the growth cone is sufficient to trigger growth cone turning toward the lower RhoA activity side.  The possible mechanism is that, when there is an asymmetry of RhoA activity in the growth cone, the filopodia at the lower RhoA activity side will extend faster then the other side because of lower inhibition or retraction, and hence the growth cone will turn towards this side.  Although we have not a tool to directly modulate the cellular Cdc42/Rac activity at present, nevertheless, from the abolish of BDNF-triggered attraction by either DN-Cdc42 and CA-Cdc42, we propose that an intracellular Cdc42 activity gradient may also be sufficient to determine the turning direction, say, filopodia extend preferentially at the higher Cdc42 activity side, and the growth cone turn towards this direction in the hence.  Further pharmaceutical studies showed that, Y-27632 and ML-7, two drugs that are capable of inhibiting myosin acticity, can block chemorepulsion triggered by gradient of either LPA or high dose ryanodine or BDNF (in the presence of PKA inhibitor), although they have no influence on chemoattraction.  This implies a key role played by myosin contractility during chemorepulsion.  Since the myosin activity can be up-regulated by both Cdc42 and RhoA, these two Rho GTPases can thus crosstalk through their convergent effect on myosin.  Our data also gave strong support to the hypothesis that asymmetric filopodial elongation determines the growth cone turning.  One important future direction is to discover how the extracellular and intracellular signals regulate different Rho GTPases, dictating the growth cone to choose which pathway, and resulting in what kind of turning.

Our knowledge about the molecular and cellular mechanisms for axon pathfinding has been greatly enlarged during the past ten years.  However, many mysteries remain to be disclosed.  One point that deserves special attention is that, many common events may happen both in axon growth and guidance during brain development, and in the regeneration of the functional nerve circuits after nerve injury.  It is one of the most difficult problems for centuries in clinical medicine to overcome the regeneration defects after nerve injury.  Every day, many unfortunate persons suffer nerve injury from various accidents, resulting in paralysis of the bodies or dysfunction of the nervous system.  Studies in axon guidance mechanism will boost the discovery of new methods in the therapy of nervous system injuries.