Essential Role of Neural Wiskott-Aldrich Syndrome Protein in Neurite Extension in PC12 Cells and Rat Hippocampal Primary Culture Cells

Abstract

Neural Wiskott-Aldrich syndrome protein (N-WASP) is an actin-regulating protein that induces filopodium formation downstream of Cdc42. It has been shown that filopodia actively extend from the growth cone, a guidance apparatus located at the tip of neurites, suggesting their role in neurite extension. Here we examined the possible involvement of N-WASP in the neurite extension process. Since verprolin, cofilin homology and acidic region (VCA) of N-WASP is known to be required for the activation of Arp2/3 complex that induces actin polymerization, we prepared a mutant (⌬cof) lacking four amino acid residues in the cofilin homology region. The corresponding residues in WASP had been reported to be mutated in some Wiskott-Aldrich syndrome patients. Expression of ⌬cof N-WASP suppressed neurite extension of PC12 cells. In support of this, the VCA region of ⌬cof cannot activate Arp2/3 complex enough compared with wild-type VCA. Furthermore, H208D mutant, which has been shown unable to bind to Cdc42, also works as a dominant negative mutant in neurite extension assay. Interestingly, the expression of H208D-⌬cof double mutant has no significant dominant negative effect. Finally, the expression of the ⌬cof mutant also severely inhibited the neurite extension of primary neurons from rat hippocampus. Thus, N-WASP is thought to be a general regulator of the actin cytoskeleton indispensable for neurite extension, which is probably caused through Cdc42 signaling and Arp2/3 complex-induced actin polymerization.The actin cytoskeleton plays a critical role in the regulation of cellular morphological change in response to various external stimuli (1, 2). In the case of neural development, actin filaments have been shown to accumulate at the growth cone (3), a guidance apparatus located at the tip of growing neurites. It has long been suggested that the actin cytoskeletal reorganization at growth cones including filopodium and lammelipodium formation is the key determinant of the direction and/or speed of neurite extension. Thus, clarification of the regulatory mechanism behind the reorganization of the actin cytoskeleton in neurons will ultimately lead to a better understanding of neural development.Evidence has been accumulating that Rho family small GTPases regulate the reorganization of the actin cytoskeleton (4). Indeed, two family members, Cdc42 and Rac, are shown to induce filopodium and lammelipodium formations, respectively (5-7). Furthermore, recent reports demonstrated that their function is essential for neurite extension in 9). However, the target proteins that function in neurite extension in neurons have yet to be identified.We found N-WASP as a 65-kDa protein that binds to the SH3 domains of Ash/Grb2 adaptor protein (10). N-WASP possesses a GBD/CRIB motif through which N-WASP directly binds to activated Cdc42 (11). In addition, N-WASP can induce filopodium formation downstream of Cdc42 in COS 7 cells (11). We have also shown that verprolin homology domain (V), which is an actin-binding site, is essential for filopo...

Regulation of Neuronal Morphology by Toca-1, an F-BAR/EFC Protein That Induces Plasma Membrane Invagination

“…The F-BAR/EFC domain induces plasma membrane invagination (8), suggesting an implication of membrane trafficking in neurite elongation. Surprisingly, knockdown of N-WASP also enhances neurite elongation in PC12 cells, which is in clear contrast to the previous report using dominant negative mutants of N-WASP (5). Meanwhile, knockdown of Toca-1 in cultured rat hippocampal neurons enhances axon branching a little but not axon elongation, whereas knockdown of N-WASP enhances both axon elongation and branching.…”

“…It has also been reported that another putative dominant negative mutant of N-WASP, which has a mutation in the GBD and is unable to bind to Cdc42, also suppresses neurite extension in PC12 cells (5).…”

“…impaired in its ability to activate Arp2/3 complex-mediated actin nucleation, reportedly suppresses neurite extension in PC12 cells and primary cultured rat hippocampal neurons (5), by working as a putative dominant negative mutant. It has also been reported that another putative dominant negative mutant of N-WASP, which has a mutation in the GBD and is unable to bind to Cdc42, also suppresses neurite extension in PC12 cells (5).…”

Signaling mechanisms that regulate actin‐based motility processes in the nervous system

“…p34Arc and p21Arc are concentrated in the actin filaments of NGF-stimulated growth cones from rat sympathetic neurons and PC12 cells (Goldberg et al 2000). Mutations in the VCA region of N-WASP block NGF-stimulated neurite growth in PC12 cells and hippocampal neurons (Banzai et al 2000), and an N-WASP mutant incapable of binding cdc42 also blocks neurite growth (Banzai et al 2000). The Drosophila version of Scar has a role in axonal development.…”

The Sorting Nexin, DSH3PX1, Connects the Axonal Guidance Receptor, Dscam, to the Actin Cytoskeleton

“…Recently Nck and phosphatidylinositol 4,5-bisphosphate have been shown to synergistically activate actin polymerization through N-Wasp in an in vitro actin pyrene assay (18). N-Wasp has also been shown to be essential in nerve growth factorstimulated neurite extension in PC12 cells, presumably through Arp2/3 complex-induced actin polymerization (19). Wasp's ability to promote the polymerization of actin has also been implicated in endocytosis (20).…”