Inclusions containing actin-depolymerizing factor (ADF) and cofilin, abundant proteins in adult human brain, are prominent in hippocampal and cortical neurites of the post-mortem brains of Alzheimer's patients, especially in neurites contacting amyloid deposits. The origin and role of these inclusions in neurodegeneration are, however, unknown. Here we show that mediators of neurodegeneration induce the rapid formation of transient or persistent rod-like inclusions containing ADF/cofilin and actin in axons and dendrites of cultured hippocampal neurons. Rods form spontaneously within neurons overexpressing active ADF/cofilin, suggesting that the activation (by dephosphorylation) of ADF/cofilin that occurs in response to neurodegenerative stimuli is sufficient to induce rod formation. Persistent rods that span the diameter of the neurite disrupt microtubules and cause degeneration of the distal neurite without killing the neuron. These findings suggest a common pathway that can lead to loss of synapses.

The actin assembly‐regulating activity of actin depolymerizing factor (ADF)/cofilin is inhibited by phosphorylation. Studies were undertaken to characterize the signaling pathways and phosphatases involved in activating phosphorylated ADF (pADF), emphasizing signals related to neuronal process extension. Western blots using antibodies to ADF and cofilin, as well as an ADF/cofilin phosphoepitope‐specific antibody characterized in this paper, were used to measure changes in the phosphorylation state and phosphate turnover of ADF/cofilin in response to inhibitors and agents known to influence growth cone motility. Increases in both [Ca2+]i and cAMP levels induced rapid pADF dephosphorylation in HT4 and cortical neurons. Calcium‐dependent dephosphorylation depended on the activation of protein phosphatase 2B (PP2B), while cAMP‐dependent dephosphorylation was likely through activation of PP1. Growth factors such as NGF and insulin also induced rapid pADF/pcofilin dephosphorylation, with NGF‐stimulated dephosphorylation in PC12 cells correlated with the translocation of ADF/cofilin to ruffling membranes. Of special interest was the finding that the rate of phosphate turnover on both pADF and pcofilin could be enhanced by growth factors without changing net pADF levels, demonstrating that growth factors can activate bifurcating pathways that promote both phosphorylation and dephosphorylation of ADF/cofilin. All experimental results indicated that dynamics of phosphorylation on ADF and cofilin are coordinately regulated. Signals that decreased pADF levels are associated with increased process extension, while agents that increased pADF levels, such as lysophosphatidic acid, inhibit process extension. These data indicate that dephosphorylation/activation of pADF is a significant response to the activation of signal pathways that regulate actin dynamics and alter cell morphology and neuronal outgrowth. Cell Motil. Cytoskeleton 39:172–190, 1998. © 1998 Wiley‐Liss, Inc.

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Growth cone motility is regulated by changes in actin dynamics. Actin depolymerizing factor (ADF) is an important regulator of actin dynamics, and extracellular signal-induced changes in ADF activity may influence growth cone motility and neurite extension. To determine this directly, we overexpressed ADF in primary neurons and analyzed neurite lengths. Recombinant adenoviruses were constructed that express wild-type Xenopus ADF/cofilin [XAC(wt)], as well as two mutant forms of XAC, the active but nonphosphorylatable XAC(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 cultured rat cortical neurons. Peak expression was approximately 75% that of endogenous ADF. XAC(wt) expression caused a slight increase in growth cone area and filopodia but decreased filopodia numbers on neurite shafts. At maximal XAC levels, neurite lengths increased >50% compared with controls infected 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 expression of the XAC(A3) mutant increased neurite extension but to a lesser extent than XAC(wt), which was partially phosphorylated. XAC expression had minimal, if any, impact on F-actin levels and did not result in compensatory changes in the expression of endogenous ADF or actin. However, F-actin turnover appeared to increase based on F-actin loss after treatment with drugs that block actin polymerization. These results provide direct evidence that increased ADF activity promotes process extension and neurite outgrowth.

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Growth cone motility and navigation in response to extracellular signals are regulated by actin dynamics. To better understand actin involvement in these processes we determined how and in what form actin reaches growth cones, and once there, how actin assembly is regulated. A continuous supply of actin is maintained at the axon tip by slow transport, the mobile component consisting of an unassembled form of actin. Actin is co-transported with actin-binding proteins, including ADF and cofilin, structurally related proteins essential for rapid turnover of actin filaments in vivo. ADF and cofilin activity is regulated through phosphorylation by LIM kinases, downstream effectors of the Rho family of GTPases, Cdc42, Rac and Rho. Attractive and repulsive extracellular guidance cues might locally alter actin dynamics by binding specific GTPase-linked receptors, activating LIM kinases, and subsequently modulating the activity of ADF/cofilin. ADF is enriched in growth cones and is required for neurite outgrowth. In addition, signals that influence growth cone behavior alter ADF/cofilin phosphorylation, and overexpression of ADF enhances neurite outgrowth. Growth promoting effects of laminin are mimicked by expression of constitutively active Cdc42 and blocked by expression of the dominant negative Cdc42. Repulsive effects of myelin and sema3D on growth cones are blocked by expression of constitutively active Rac1 and dominant negative Rac1, respectively. Thus a series of complex pathways must exist for regulating effectors of actin dynamics. The bifurcating nature of the ADF/cofilin phosphorylation pathway may provide the integration necessary for this complex regulation.