ßPix-d promotes tubulin acetylation and neurite outgrowth through a PAK/Stathmin1 signaling pathway.

Microtubules are a major cytoskeletal component of neurites, and the regulation of microtubule stability is essential for neurite morphogenesis. ßPix (ARHGEF7) is a guanine nucleotide exchange factor for the small GTPases Rac1 and Cdc42, which modulate the organization of actin filaments and microtubules. ßPix is expressed as alternatively spliced variants, including the ubiquitous isoform ßPix-a and the neuronal isoforms ßPix-b and ßPix-d, but the function of the neuronal isoforms remains unclear. Here, we reveal the novel role of ßPix neuronal isoforms in regulating tubulin acetylation and neurite outgrowth. At DIV4, hippocampal neurons cultured from ßPix neuronal isoform knockout (ßPix-NIKO) mice exhibit defects in neurite morphology and tubulin acetylation, a type of tubulin modification which often labels stable microtubules. Treating ßPix-NIKO neurons with paclitaxel, which stabilizes the microtubules, or reintroducing either neuronal ßPix isoform to the KO neurons overcomes the impairment in neurite morphology and tubulin acetylation, suggesting that neuronal ßPix isoforms may promote microtubule stabilization during neurite development. ßPix-NIKO neurons also exhibit lower phosphorylation levels for Stathmin1, a microtubule-destabilizing protein, at Ser16. Expressing either ßPix neuronal isoform in the ßPix-NIKO neurons restores Stathmin1 phosphorylation levels, with ßPix-d having a greater effect than ßPix-b. Furthermore, we find that the recovery of neurite length and Stathmin1 phosphorylation via ßPix-d expression requires PAK kinase activity. Taken together, our study demonstrates that ßPix-d regulates the phosphorylation of Stathmin1 in a PAK-dependent manner and that neuronal ßPix isoforms promote tubulin acetylation and neurite morphogenesis during neuronal development.