Fig. 2.

Dkk1 synaptotoxicity is Daam1 dependent. (A) Schematic of the Wnt-PCP pathway, showing the two arms branching below disheveled, acting via Daam/Rho/ROCK to regulate cytoskeletal dynamics and JNK/c-Jun primarily to regulate gene transcription. (B) Primary cortical neuronal cultures were treated with DAAM1 or DAAM2 Pen-1–coupled siRNA duplexes for 48 hours, harvested, and analyzed by Western blotting for Daam1 and Daam2. Daam1 was detectable in untreated cells, whereas Daam2 was not. Daam1-si potently reduced Daam1 protein expression levels. (C and D) Cultures were treated overnight with DAAM1- or DAAM2-siRNA duplexes. Next day, cells were treated with 400 ng/mL recombinant Dkk1 protein for 3 hours, fixed, and fluorescently labeled with phalloidin-488, imaged (C), scale bar = 50 μM, and F-actin–labeled puncta quantified (D). Significance determined by ANOVA and Tukey's post hoc t-test. Error bars indicate standard deviation. Abbreviations: Aβ, amyloid β; ANOVA, analysis of variance; c-Abl1, c-Abl oncogene 1, nonreceptor tyrosine kinase (ABL1); d.i.v., days in vitro; DAAM1, disheveled associated activator of morphogenesis 1; Dkk1, Dickkopf-1; Dvl, disheveled; EGR1, early growth response 1; Fzd, frizzled; GSK3-α/β, glycogen synthase kinase-α/β; JNK1, c-Jun N-terminal kinase (MAPK8); KLF10, Krüppel-like factor 10; LRP6, low-density lipoprotein receptor–related protein 6; MKK4/7, mitogen-activated protein kinase 4/7 (MAP2K4 and MAP2K7); NAB2, NGFI-A binding protein 2; PCP, planar cell polarity; RhoA, Ras homolog family member A; ROCK, Rho-associated coiled-coil containing protein kinase; Vangl2, Van Gogh–like protein 2.