Increase in neurite outgrowth mediated by overexpression of actin depolymerizing factor

Growth cone motility is regulated by changes in actin dynamics. Actin depol ymerizing factor (ADF) is an important regulator of actin dynamics, and ext racellular signal-induced changes in ADF activity may influence growth cone motility and neurite extension. To determine this directly, we overexpress ed ADF in primary neurons and analyzed neurite lengths. Recombinant adenovi ruses were constructed that express wild-type Xenopus ADF/cofilin [XAC(wt)] , as well as two mutant forms of XAC, the active but nonphosphorylatable XA C(A3) and the less active, pseudophosphorylated XAC(E3). XAC expression was detectable on Western blots 24 hr after infection and peaked at 3 d in cul tured rat cortical neurons. Peak expression was similar to 75% that of endo genous ADF. XAC(wt) expression caused a slight increase in growth cone area and filopodia but decreased filopodia numbers on neurite shafts. At maxima l XAC levels, neurite lengths increased >50% compared with controls infecte d with a green fluorescent protein-expressing adenovirus. Increased neurite extension was directly related to the expression of active XAC. Expression of the XAC(E3) mutant did not increase neurite extension, whereas expressi on of the XAC(A3) mutant increased neurite extension but to a lesser extent than XAC(wt), which was partially phosphorylated. XAC expression had minim al, if any, impact on F-actin levels and did not result in compensatory cha nges in the expression of endogenous ADF or actin. However, F-actin turnove r appeared to increase based on F-actin loss after treatment with drugs tha t block actin polymerization. These results provide direct evidence that in creased ADF activity promotes process extension and neurite outgrowth.