Valproic acid-inducible Arl4D and cytohesin-2/ARNO, acting through the downstream Arf6, regulate neurite outgrowth in N1E-115 cells

Exp Cell Res. 2009 Jul 15;315(12):2043-52. doi: 10.1016/j.yexcr.2009.03.012. Epub 2009 Mar 25.


The mood-stabilizing agent valproic acid (VPA) potently promotes neuronal differentiation. As yet, however, little is known about the underlying molecular mechanism. Here, we show that VPA upregulates cytohesin-2 and mediates neurite outgrowth in N1E-115 neuroblastoma cells. Cytohesin-2 is the guanine-nucleotide exchange factor (GEF) for small GTPases of the Arf family; it regulates many aspects of cellular functions including morphological changes. Treatment with the specific cytohesin family inhibitor SecinH3 or knockdown of cytohesin-2 with its siRNA results in blunted induction of neurite outgrowth in N1E-115 cells. The outgrowth is specifically inhibited by siRNA knockdown of Arf6, but not by that of Arf1. Furthermore, VPA upregulates Arl4D, an Arf-like small GTPase that has recently been identified as the regulator that binds to cytohesin-2. Arl4D knockdown displays an inhibitory effect on neurite outgrowth resulting from VPA, while expression of constitutively active Arl4D induces outgrowth. We also demonstrate that the addition of cell-permeable peptide, coupling the cytohesin-2-binding region of Arl4D into cells, reduces the effect of VPA. Thus, Arl4D is a previously unknown regulator of neurite formation through cytohesin-2 and Arf6, providing another example that the functional interaction of two different small GTPases controls an important cellular function.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ADP-Ribosylation Factor 6
  • ADP-Ribosylation Factors / biosynthesis
  • ADP-Ribosylation Factors / physiology*
  • Animals
  • Anticonvulsants / pharmacology*
  • Cell Line
  • Cell Line, Tumor
  • GTPase-Activating Proteins / physiology*
  • Mice
  • Neurites / drug effects*
  • Neurites / physiology
  • Neurogenesis
  • Triazoles / pharmacology
  • Up-Regulation
  • Valproic Acid / pharmacology*


  • ADP-Ribosylation Factor 6
  • Anticonvulsants
  • GTPase-Activating Proteins
  • SecinH3
  • Triazoles
  • cytohesin-2
  • Valproic Acid
  • ADP-Ribosylation Factors
  • Arf6 protein, mouse
  • Arl5a protein, mouse