December 5, 2018
Synaptosomal protein fractions (P2C) corresponding to 20 mg protein were dissolved in lysis buffer and processed according to a filter-aided sample preparation (FASP) protocol modified essentially as described by Distler et al. Unless stated otherwise, all steps were automated on a liquid-handling workstation equipped with a vacuum manifold (Freedom EVO 150, Tecan) by using an adaptor device constructed in-house
The establishment of axon-dendrite polarity is fundamental for radial migration of neurons during cortex development of mammals. We demonstrate that the E3 ubiquitin ligases WW-Containing Proteins 1 and 2 (Wwp1 and Wwp2) are indispensable for proper polarization of developing neurons. We show that knockout of Wwp1 and Wwp2 results in defects in axon-dendrite polarity in pyramidal neurons, and their aberrant laminar cortical distribution. Knockout of miR-140, encoded in Wwp2 intron, engenders phenotypic changes analogous to those upon Wwp1 and Wwp2 deletion. Intriguingly, transcription of the Wwp1 and Wwp2/miR-140 loci in neurons is induced by the transcription factor Sox9. Finally, we provide evidence that miR-140 supervises the establishment of axon-dendrite polarity through repression of Fyn kinase mRNA. Our data delineate a novel regulatory pathway that involves Sox9-[Wwp1/Wwp2/miR-140]-Fyn required for axon specification, acquisition of pyramidal morphology, and proper laminar distribution of cortical neurons. Copyright Š 2018 Elsevier Inc. All rights reserved.
Ambrozkiewicz, MC; Schwark, M; Kishimoto-Suga, M; Borisova, E; Hori, K; Salazar-LĂĄzaro, A; Rusanova, A; Altas, B; Piepkorn, L; Bessa, P; Schaub, T; Zhang, X; Rabe, T; Ripamonti, S; RosĂĄrio, M; Akiyama, H; Jahn, O; Kobayashi, T; Hoshino, M; Tarabykin, V; Kawabe, H;
Journal: Neuron Pages: 1097-1115.e15