The results reported here support a role for ARF6 and its downstream lipid-modifying effectors in the controlled extension and branching of axonal processes. These effects of ARNO and ARF6 upon axonal growth are distinct from our previous findings in dendrites (Hernandez-Deviez et al., 2002
), in that the enhancement of axonal length and branching caused by expression of mutant ARF6 and ARNO are mediated by the PI(4)P 5-Kinase α pathway rather than through the small GTPase Rac1. These effects may be through changes in the dynamics of the actin cytoskeleton, because expression of inactive ARNO or ARF6 resulted in a specific depletion of the actin-binding protein Mena from the growth cone. These data suggest that, during development, ARNO and ARF6 play an important role in the regulation of axonal elongation and sprouting through effects upon membrane structure and cytoskeleton dynamics.
During axonal development three distinct events occur: elongation, pathfinding, and branching. Rho GTPases have been implicated in the regulation of many aspects of axonal development (Luo, 2000
) because progressive deletion of the three Drosophila
Rac GTPases result in differential defects in axon outgrowth, guidance, and branching (Ng et al., 2002
). We found that catalytically inactive ARNO and ARF6 caused both an increase in axonal length and in axonal collateral branches. Although ARF6 and Rac1 seem to act in concert to modulate dendritic development in hippocampal neurons (Hernandez-Deviez et al., 2002
), our results suggest that Rac1 is not downstream of ARF6 in axonal extension and branching and must reflect an additional regulatory pathway. This contrasts with recent reports showing that neurite extension in chick retinal cells and Aplysia
motor neurons is activated by ARF6 (Huh et al., 2003
) and ARF6/PIX/p95-APP1 (Albertinazzi et al., 2003
). Whether these discrepancies reflect differences in cell type or in the regulation of axonal elongation remains unclear.
In hippocampal neurons, it seems that ARF6 modulates axonal extension and branching through effects upon membrane structure. ARF6 stimulates PI(4)P 5-Kinase (Honda et al., 1999
) and PIP2
has been shown to be enriched at sites of ARF6 activation (Brown et al., 2001
It has recently been shown that dominant negative type I PI(4)P 5-Kinase α promotes neurite elongation in neuroblastoma cells, suggesting that PI(4,5)P2
negatively regulates neuritogenesis (van Horck et al., 2002
; Yamazaki et al., 2002
). We found that coexpression of type I PI(4)P 5-Kinase α and catalytically inactive ARNO or dominant negative ARF6 dramatically reversed the increased axonal length and branching of cells expressing inactive ARNO alone, indicating that PI(4)P 5-Kinase α is downstream of ARF6 in regulating axonal extension.
In neurons expressing catalytically inactive ARNO, Mena, but not other cytoskeletal-associated proteins, was depleted from the growth cone and expression of constitutively active ARF6 or PI(4)P 5-Kinase resulted in reassociation of Mena with the growth cone filopodia and lamellipodia. Although Ena/VASP proteins mediate actin polymerization (Laurent et al., 1999
) and have been shown to be localized to the leading edge of migrating cells and at growth cone tips (Gertler et al., 1996
), recent studies have shown that depletion of Mena from the plasma membrane promotes fibroblast motility (Bear et al., 2000
). This apparent paradox is incompletely understood, but recent studies suggest that Mena may act to control actin filament length by acting as an “anticapping” molecule at the barbed ends, resulting in longer, more flexible filaments (Bear et al., 2002
; Krause et al., 2002
). Because extension of lamellipodia seems to require short filaments in highly branched networks (Bear et al., 2002
), antagonizing Mena should result in increased extension of the leading edge. In fact, incubation of neurons with low concentrations of the barbed-end capping drug cytochalasin B or D causes depletion of Mena from the growth cone and increased neurite growth (Marsh and Letourneau, 1984
; Bear et al., 2002
). Because ARF6 activation of PI(4)P 5-Kinase increases PIP2
levels, this would inhibit gelsolin, inducing uncapping of actin filaments, resulting in free barbed ends and subsequently Mena localization at the plasma membrane. The ultimate consequence of this increased PIP2
would be to restrict growth cone motility and axonal extension. Further ultrastructural analysis of the actin filaments awaits additional investigation.
ARNO and ARF6 play an essential role in both actin dynamics and membrane traffic in various cell types (Radhakrishna et al., 1996
; D'Souza-Schorey et al., 1997
; Radhakrishna and Donaldson, 1997
; Frank et al., 1998b
; Radhakrishna et al., 1999
; Boshans et al., 2000
; Palacios et al., 2001
) and ARF6, through phosphatidylinositol kinases, has been implicated in the regulation of synaptic vesicle recycling (Krauss et al., 2003
) and regulated exocytosis (Aikawa and Martin, 2003
). Here, we have presented data that suggest that ARF6 activation regulates axonal elongation and branching through modulation of the growth cone cytoskeleton. However, we have also found that expression of inactive ARNO causes a redistribution of specialized endosomes to the axonal but not dendritic plasma membrane (Hernández-Deviez and Wilson, unpublished data), suggesting that ARNO regulates membrane cycling in neurons and may also control axonal elongation and branching by increasing membrane available for growth.
Axon regeneration after injury is blocked by various growth-inhibitory proteins, and efforts have been made to develop strategies to promote axonal regrowth in injured adult neurons. Small GTPases are common targets of many of these inhibitors (Fitch and Silver, 1997
; Fournier and Strittmatter, 2001
). Recently, it has been found that injured axons regrow on inhibitory substrates when Rho GTPase is inactivated (Lehmann et al., 1999
). In addition, pharmacological inhibition of its major downstream effector, Rhokinase, promotes axonal outgrowth (Borisoff et al., 2003
; Fournier et al., 2003
). These findings suggest that small GTPases as well as their effectors constitute potential targets to disrupt inhibitory signaling. Our study suggests that manipulation of the active state of ARNO, ARF6, and PI(4)P 5-Kinase are potential targets to promote axonal regeneration after injury.