Neurodevelopmental disorder-associated CYFIP2 regulates membraneless organelles and eIF2α phosphorylation via protein interactors and actin cytoskeleton.

De novo variants in the Cytoplasmic FMR1-interacting protein 2 (CYFIP2) have been repeatedly associated with neurodevelopmental disorders and epilepsy, underscoring its critical role in brain development and function. While CYFIP2's role in regulating actin polymerization as part of the WAVE regulatory complex (WRC) is well-established, its additional molecular functions remain relatively unexplored. In this study, we performed unbiased quantitative proteomic analysis, revealing 278 differentially expressed proteins (DEPs) in the forebrain of Cyfip2 knock-out embryonic mice compared to wild-type mice. Unexpectedly, these DEPs, in conjunction with previously identified CYFIP2 brain interactors, included not only other WRC components but also numerous proteins associated with membraneless organelles (MLOs) involved in mRNA processing and translation within cells, including the nucleolus, stress granules, and processing bodies. Additionally, single-cell transcriptomic analysis of the Cyfip2 knock-out forebrain revealed gene expression changes linked to cellular stress responses and MLOs. We also observed morphological changes in MLOs in Cyfip2 knock-out brains and CYFIP2 knock-down cells under basal and stress conditions. Lastly, we demonstrated that CYFIP2 knock-down in cells, potentially through WRC-dependent actin regulation, suppressed the phosphorylation levels of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α), thereby enhancing protein synthesis. These results suggest a physical and functional connection between CYFIP2 and various MLO proteins and also extend CYFIP2's role within the WRC from actin regulation to influencing eIF2α phosphorylation and protein synthesis. With these dual functions, CYFIP2 may fine-tune the balance between MLO formation/dynamics and protein synthesis, a crucial aspect of proper mRNA processing and translation.

An N-terminal and ankyrin repeat domain interactome of Shank3 identifies the protein complex with the splicing regulator Nono in mice.

An autism-associated gene Shank3 encodes multiple splicing isoforms, Shank3a-f. We have recently reported that Shank3a/b-knockout mice were more susceptible to kainic acid-induced seizures than wild-type mice at 4 weeks of age. Little is known, however, about how the N-terminal and ankyrin repeat domains (NT-Ank) of Shank3a/b regulate multiple molecular signals in the developing brain. To explore the functional roles of Shank3a/b, we performed a mass spectrometry-based proteomic search for proteins interacting with GFP-tagged NT-Ank. In this study, NT-Ank was predicted to form a variety of complexes with a total of 348 proteins, in which RNA-binding (n = 102), spliceosome (n = 22), and ribosome-associated molecules (n = 9) were significantly enriched. Among them, an X-linked intellectual disability-associated protein, Nono, was identified as a NT-Ank-binding protein. Coimmunoprecipitation assays validated the interaction of Shank3 with Nono in the mouse brain. In agreement with these data, the thalamus of Shank3a/b-knockout mice aberrantly expressed splicing isoforms of autism-associated genes, Nrxn1 and Eif4G1, before and after seizures with kainic acid treatment. These data indicate that Shank3 interacts with multiple RNA-binding proteins in the postnatal brain, thereby regulating the homeostatic expression of splicing isoforms for autism-associated genes after birth.

Cell-autonomous reduction of CYFIP2 changes dendrite length, dendritic protrusion morphology, and inhibitory synapse density in the hippocampal CA1 pyramidal neurons of 17-month-old mice.

The cytoplasmic FMR1-interacting protein 2 (CYFIP2) have diverse molecular functions in neurons, including the regulation of actin polymerization, mRNA translation, and mitochondrial morphology and function. Mutations in the CYFIP2 gene are associated with early-onset epilepsy and neurodevelopmental disorders, while decreases in its protein levels are linked to Alzheimer's disease (AD). Notably, previous research has revealed AD-like phenotypes, such as dendritic spine loss, in the hippocampal CA1 pyramidal neurons of 12-month-old Cyfip2 heterozygous mice but not of age-matched CA1 pyramidal neuron-specific Cyfip2 conditional knock-out (cKO) mice. This study aims to investigate whether dendritic spine loss in Cyfip2 cKO mice is merely delayed compared to Cyfip2 heterozygous mice, and to explore further neuronal phenotypes regulated by CYFIP2 in aged mice. We characterized dendrite and dendritic protrusion morphologies, along with excitatory/inhibitory synapse densities in CA1 pyramidal neurons of 17-month-old Cyfip2 cKO mice. Overall dendritic branching was normal, with a reduction in the length of basal, not apical, dendrites in CA1 pyramidal neurons of Cyfip2 cKO mice. Furthermore, while dendritic protrusion density remained normal, alterations were observed in the length of mushroom spines and the head volume of stubby spines in basal, not apical, dendrites of Cyfip2 cKO mice. Although excitatory synapse density remained unchanged, inhibitory synapse density increased in apical, not basal, dendrites of Cyfip2 cKO mice. Consequently, a cell-autonomous reduction of CYFIP2 appears insufficient to induce dendritic spine loss in CA1 pyramidal neurons of aged mice. However, CYFIP2 is required to maintain normal dendritic length, dendritic protrusion morphology, and inhibitory synapse density.