Imbalance between RNA-binding proteins and the WAVE regulatory complex underlies the toxic gain-of-function effects of the CYFIP2 Arg87Cys variant in neurodevelopmental disorders

Pathogenic variants in cytoplasmic FMR1-interacting protein 2 (CYFIP2) cause neurodevelopmental disorders, with hotspot substitutions at arginine 87 (Arg87) associated with particularly severe symptoms. CYFIP2 functions both as a component of the WAVE (Wiskott-Aldrich syndrome protein family verprolin-homologous protein) regulatory complex (WRC), regulating the actin cytoskeleton, and as an interactor of RNA-binding proteins (RBPs) involved in mRNA processing/translation. Although Arg87 variants reduce CYFIP2 protein stability, heterozygous knock-in mice carrying Arg87Cys (Cyfip2+/R87C) show markedly more severe phenotypes than heterozygous knockout (Cyfip2+/-) mice, supporting a toxic gain-of-function mechanism. However, how the dual roles of CYFIP2 contribute to this mechanism remains unclear. To address this, we examined Cyfip2+/R87C mice at postnatal week 7, before overt neuronal loss and gliosis, focusing on the progressive dispersion of hippocampal cornu ammonis 1 (CA1) pyramidal neurons, a phenotype absent in Cyfip2+/- mice. Single-nucleus RNA sequencing suggested candidate alterations related to translation and cell adhesion/migration in CA1 pyramidal neurons. Complementarily, human embryonic kidney 293T cells expressing CYFIP2 Arg87Cys showed defects in proliferation and morphology, accompanied by altered expression of cell adhesion-related genes. Quantitative proteomic analysis of the CA1 region uncovered an imbalance between CYFIP2-associated protein networks, characterized by increased RBPs and reduced WRC components. Phosphoproteomic analysis further revealed altered phosphorylation of synaptic and translational regulators. Consistently, whole-cell recordings and biocytin-based morphological analyses showed increased frequency of miniature excitatory postsynaptic currents and altered dendritic morphology in Cyfip2+/R87C CA1 pyramidal neurons, most prominently in the more severely dispersed deep-layer neurons. Moreover, the cell adhesion molecule Eph receptor A3 was reduced, possibly as a result of RBP dysregulation. Together, these findings suggest that an imbalance between RBP- and WRC-associated pathways underlies the gain-of-function effects of CYFIP2 Arg87Cys and contributes to early neuronal phenotypes, most notably CA1 layer dispersion. This mechanism, distinct from CYFIP2 haploinsufficiency, supports allele-specific therapeutic strategies for Arg87 variants.