Twinfilin-1 phosphorylation in reelin signaling regulates actin dynamics and spine development

ABSTRACT Reelin is an extracellular glycoprotein essential for neuronal migration, spine development, and synaptic plasticity. Impaired reelin signaling is linked to neurological disorders, including schizophrenia and autism. While reelin mutant (reeler) mice exhibit behavioral deficits associated with impaired spine formation, the underlying molecular mechanisms remain unclear. We identified Twinfilin-1 (Twf1) as a downstream effector of reelin signaling via phosphoproteomic analysis, based on its reduced tyrosine phosphorylation in reeler mice. We found that Src regulated Twf1 phosphorylation at tyrosine 309, and reelin stimulation increased Twf1 phosphorylation in neurons, an effect blocked by the Src inhibitor PP2. A phospho-resistant Twf1 mutant (Twf1 Y309F) showed reduced capping protein binding and a lower F/G-actin ratio. Twf1Y309F mice exhibited cognitive deficits, reduced spine density, smaller spine head size, and a decreased F/G-actin ratio in synaptosomes. These findings highlight Twf1 phosphorylation as a key component of reelin signaling involved in actin remodeling and spine development. Competing Interest Statement The authors have declared no competing interest. Abbreviations - Twf1 - Twinfilin-1 - ADF-H - actin-depolymerizing factor homology - RELN - reelin - ApoER2 - apolipoprotein E receptor 2 - VLDLR - very low-density lipoprotein receptor - CP - capping protein - Dab1 - Disabled homolog 1 - DIV - days in vitro - SFKs - Src family kinases - Src - proto-oncogene tyrosine-protein kinase Src - Fyn - proto-oncogene tyrosine-protein kinase Fyn - HEK293T - human embryonic kidney 293T cells - hTWF1 - human Twinfilin-1 - iPSC - induced pluripotent stem cell - CA - constitutively active - KD - kinase-dead - LC-MS/MS - liquid chromatography–tandem mass spectrometry - mPFC - medial prefrontal cortex - pTwf1 - phosphorylated Twf1 - pY - phosphotyrosine - PFA - paraformaldehyde - VD - visual discrimination - RL - reversal learning - WT - wild-type - Y309 - tyrosine 309.