CYFIP2 p.Arg87Cys Causes Neurological Defects and Degradation of CYFIP2.

Here, we report the generation and comprehensive characterization of a knockin mouse model for the hotspot p.Arg87Cys variant of the cytoplasmic FMR1-interacting protein 2 (CYFIP2) gene, which was recently identified in individuals diagnosed with West syndrome, a developmental and epileptic encephalopathy. The Cyfip2+/R87C mice recapitulated many neurological and neurobehavioral phenotypes of the patients, including spasmlike movements, microcephaly, and impaired social communication. Age-progressive cytoarchitectural disorganization and gliosis were also identified in the hippocampus of Cyfip2+/R87C mice. Beyond identifying a decrease in CYFIP2 protein levels in the Cyfip2+/R87C brains, we demonstrated that the p.Arg87Cys variant enhances ubiquitination and proteasomal degradation of CYFIP2. ANN NEUROL 2023;93:155-163.

Protein interactome and cell-type expression analyses reveal that cytoplasmic FMR1-interacting protein 1 (CYFIP1), but not CYFIP2, associates with astrocytic focal adhesion.

The two members of the cytoplasmic FMR1-interacting protein family, CYFIP1 and CYFIP2, are evolutionarily conserved multifunctional proteins whose defects are associated with distinct types of brain disorders. Even with high sequence homology between CYFIP1 and CYFIP2, several lines of evidence indicate their different functions in the brain; however, the underlying mechanisms remain largely unknown. Here, we performed reciprocal immunoprecipitation experiments using CYFIP1-2 × Myc and CYFIP2-3 × Flag knock-in mice and found that CYFIP1 and CYFIP2 are not significantly co-immunoprecipitated with each other in the knock-in brains compared with negative control wild-type (WT) brains. Moreover, CYFIP1 and CYFIP2 showed different size distributions by size-exclusion chromatography of WT mouse brains. Specifically, mass spectrometry-based analysis of CYFIP1-2 × Myc knock-in brains identified 131 proteins in the CYFIP1 interactome. Comparison of the CYFIP1 interactome with the previously identified brain region- and age-matched CYFIP2 interactome, consisting of 140 proteins, revealed only eight common proteins. Investigations using single-cell RNA-sequencing databases suggested non-neuronal cell- and neuron-enriched expression of Cyfip1 and Cyfip2, respectively. At the protein level, CYFIP1 was detected in both neurons and astrocytes, while CYFIP2 was detected only in neurons, suggesting the predominant expression of CYFIP1 in astrocytes. Bioinformatic characterization of the CYFIP1 interactome, and co-expression analysis of Cyfip1 with astrocytic genes, commonly linked CYFIP1 with focal adhesion proteins. Immunocytochemical analysis and proximity ligation assay suggested partial co-localization of CYFIP1 and focal adhesion proteins in cultured astrocytes. Together, these results suggest a CYFIP1-specific association with astrocytic focal adhesion, which may contribute to the different brain functions and dysfunctions of CYFIP1 and CYFIP2. Cover Image for this issue: https://doi.org/10.1111/jnc.15410.

Epilepsy- and intellectual disability-associated CYFIP2 interacts with both actin regulators and RNA-binding proteins in the neonatal mouse forebrain.

Variants of the cytoplasmic FMR1-interacting protein 2 (CYFIP2) gene are associated with early-onset epileptic encephalopathy, intellectual disability, and developmental delay. However, the current understanding of the molecular functions of CYFIP2 is limited to those related to actin dynamics, and thus, the detailed mechanisms of CYFIP2-associated brain disorders remain largely unknown. Here, we isolated the neonatal forebrain CYFIP2 complex using newly generated Cyfip2-3×Flag knock-in mice, and performed mass spectrometry-based analyses to identify proteins in the complex. The CYFIP2 interactome, consisting of 140 proteins, contained not only the expected actin regulators but also 25 RNA-binding proteins (RBPs) including Argonaute proteins. Functionally, overexpression of brain disorder-associated CYFIP2 R87 variants, but not wild-type, inhibited stress granule formation in HeLa cells. Mechanistically, the CYFIP2 R87 variants formed intracellular clusters with Argonaute proteins under both basal and stress conditions, and thereby possibly preventing their assembly into stress granules. Beyond identifying CYFIP2 interactors in vivo, these results may provide novel insights for better understanding the molecular mechanisms of CYFIP2-associated brain disorders.

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.

Cell-autonomous reduction of CYFIP2 is insufficient to induce Alzheimer's disease-like pathologies in the hippocampal CA1 pyramidal neurons of aged mice.

Cytoplasmic FMR1-interacting protein 2 (CYFIP2) is an evolutionarily conserved multifunctional protein that regulates the neuronal actin cytoskeleton, mRNA translation and transport, and mitochondrial morphology and function. Supporting its critical roles in proper neuronal development and function, human genetic studies have repeatedly identified variants of the CYFIP2 gene in individuals diagnosed with neurodevelopmental disorders. Notably, a few recent studies have also suggested a mechanistic link between reduced CYFIP2 level and Alzheimer's disease (AD). Specifically, in the hippocampus of 12-month-old Cyfip2 heterozygous mice, several AD-like pathologies were identified, including increased levels of Tau phosphorylation and gliosis, and loss of dendritic spines in CA1 pyramidal neurons. However, detailed pathogenic mechanisms, such as cell types and their circuits where the pathologies originate, remain unknown for AD-like pathologies caused by CYFIP2 reduction. In this study, we aimed to address this issue by examining whether the cell-autonomous reduction of CYFIP2 in CA1 excitatory pyramidal neurons is sufficient to induce AD-like phenotypes in the hippocampus. We performed immunohistochemical, morphological, and biochemical analyses in 12-month-old Cyfip2 conditional knock-out mice, which have postnatally reduced CYFIP2 expression level in CA1, but not in CA3, excitatory pyramidal neurons of the hippocampus. Unexpectedly, we could not find any significant AD-like phenotype, suggesting that the CA1 excitatory neuron-specific reduction of CYFIP2 level is insufficient to lead to AD-like pathologies in the hippocampus. Therefore, we propose that CYFIP2 reduction in other neurons and/or their synaptic connections with CA1 pyramidal neurons may be critically involved in the hippocampal AD-like phenotypes of Cyfip2 heterozygous mice.

The emerging roles of Shank3 in cardiac function and dysfunction.

Shank3 is a member of the Shank family proteins (Shank1-3), which are abundantly present in the postsynaptic density (PSD) of neuronal excitatory synapses. As a core scaffold in the PSD, Shank3 plays a critical role in organizing the macromolecular complex, ensuring proper synaptic development and function. Clinically, various mutations of the SHANK3 gene are causally associated with brain disorders such as autism spectrum disorders and schizophrenia. However, recent in vitro and in vivo functional studies and expression profiling in various tissues and cell types suggest that Shank3 also plays a role in cardiac function and dysfunction. For example, Shank3 interacts with phospholipase Cß1b (PLCß1b) in cardiomyocytes, regulating its localization to the sarcolemma and its role in mediating Gq-induced signaling. In addition, changes in cardiac morphology and function associated with myocardial infarction and aging have been investigated in a few Shank3 mutant mouse models. This review highlights these results and potential underlying mechanisms, and predicts additional molecular functions of Shank3 based on its protein interactors in the PSD, which are also highly expressed and function in the heart. Finally, we provide perspectives and possible directions for future studies to better understand the roles of Shank3 in the heart.

Increased ribosomal protein levels and protein synthesis in the striatal synaptosome of Shank3-overexpressing transgenic mice.

The SH3 and multiple ankyrin repeat domains 3 (Shank3) protein is a core organizer of the macromolecular complex in excitatory postsynapses, and its defects cause numerous synaptopathies, including autism spectrum disorders. Although the function of Shank3 as a postsynaptic scaffold is adequately established, other potential mechanisms through which Shank3 broadly modulates the postsynaptic proteome remain relatively unexplored. In our previous quantitative proteomic analysis, six up-regulated ribosomal proteins were identified in the striatal synaptosome of Shank3-overexpressing transgenic (TG) mice. In the present study, we validated the increased levels of RPLP1 and RPL36A in synaptosome, but not in whole lysate, of the TG striatum. Moreover, protein synthesis and extracellular signaling-regulated kinase (ERK) activity were enhanced in the TG striatal synaptosome. To understand the potential contribution of increased protein synthesis to the proteomic change in the TG striatal synaptosome, we performed RNA-sequencing analyses on both whole synaptosomal and synaptic polysome-enriched fractions. Comparative analyses showed a positive correlation only between the polysome-associated transcriptome and up-regulated proteome in the TG striatal synaptosome. Our findings suggest a novel mechanism through which Shank3 may remodel the postsynaptic proteome by regulating synaptic protein synthesis, whose dysfunction can be implicated in SHANK3-associated synaptopathies.

Altered presynaptic function and number of mitochondria in the medial prefrontal cortex of adult Cyfip2 heterozygous mice.

Variants of the cytoplasmic FMR1-interacting protein (CYFIP) gene family, CYFIP1 and CYFIP2, are associated with numerous neurodevelopmental and neuropsychiatric disorders. According to several studies, CYFIP1 regulates the development and function of both pre- and post-synapses in neurons. Furthermore, various studies have evaluated CYFIP2 functions in the postsynaptic compartment, such as regulating dendritic spine morphology; however, no study has evaluated whether and how CYFIP2 affects presynaptic functions. To address this issue, in this study, we have focused on the presynapses of layer 5 neurons of the medial prefrontal cortex (mPFC) in adult Cyfip2 heterozygous (Cyfip2+/-) mice. Electrophysiological analyses revealed an enhancement in the presynaptic short-term plasticity induced by high-frequency stimuli in Cyfip2+/- neurons compared with wild-type neurons. Since presynaptic mitochondria play an important role in buffering presynaptic Ca2+, which is directly associated with the short-term plasticity, we analyzed presynaptic mitochondria using electron microscopic images of the mPFC. Compared with wild-type mice, the number, but not the volume or cristae density, of mitochondria in both presynaptic boutons and axonal processes in the mPFC layer 5 of Cyfip2+/- mice was reduced. Consistent with an identification of mitochondrial proteins in a previously established CYFIP2 interactome, CYFIP2 was detected in a biochemically enriched mitochondrial fraction of the mouse mPFC. Collectively, these results suggest roles for CYFIP2 in regulating presynaptic functions, which may involve presynaptic mitochondrial changes.

The Neomycin Resistance Cassette in the Targeted Allele of Shank3B Knock-Out Mice Has Potential Off-Target Effects to Produce an Unusual Shank3 Isoform.

Variants of the SH3 and multiple ankyrin repeat domains 3 (SHANK3), which encodes postsynaptic scaffolds, are associated with brain disorders. The targeted alleles in a few Shank3 knock-out (KO) lines contain a neomycin resistance (Neo) cassette, which may perturb the normal expression of neighboring genes; however, this has not been investigated in detail. We previously reported an unexpected increase in the mRNA expression of Shank3 exons 1-12 in the brains of Shank3B KO mice generated by replacing Shank3 exons 13-16 with the Neo cassette. In this study, we confirmed that the increased Shank3 mRNA in Shank3B KO brains produced an unusual ∼60 kDa Shank3 isoform (Shank3-N), which did not properly localize to the synaptic compartment. Functionally, Shank3-N overexpression altered the dendritic spine morphology in cultured neurons. Importantly, Shank3-N expression in Shank3B KO mice was not a compensatory response to a reduction of full-length Shank3 because expression was still detected in the brain after normalizing the level of full-length Shank3. Moreover, in another Shank3 KO line (Shank3 gKO) with a similar Shank3 exonal deletion as that in Shank3B KO mice but without a Neo cassette, the mRNA expression levels of Shank3 exons 1-12 were lower than those of wild-type mice and Shank3-N was not detected in the brain. In addition, the expression levels of genes neighboring Shank3 on chromosome 15 were altered in the striatum of Shank3B KO but not Shank3 gKO mice. These results suggest that the Neo cassette has potential off-target effects in Shank3B KO mice.

Enhanced Prefrontal Neuronal Activity and Social Dominance Behavior in Postnatal Forebrain Excitatory Neuron-Specific Cyfip2 Knock-Out Mice.

The cytoplasmic fragile X mental retardation 1 (FMR1)-interacting protein 2 (CYFIP2) gene is associated with epilepsy, intellectual disability (ID), and developmental delay, suggesting its critical role in proper neuronal development and function. CYFIP2 is involved in regulating cellular actin dynamics and also interacts with RNA-binding proteins. However, the adult brain function of CYFIP2 remains unclear because investigations thus far are limited to Cyfip2 heterozygous (Cyfip2+/- ) mice owing to the perinatal lethality of Cyfip2-null mice. Therefore, we generated Cyfip2 conditional knock-out (cKO) mice with reduced CYFIP2 expression in postnatal forebrain excitatory neurons (CaMKIIα-Cre). We found that in the medial prefrontal cortex (mPFC) of adult Cyfip2 cKO mice, CYFIP2 expression was decreased in both layer 2/3 (L2/3) and layer 5 (L5) neurons, unlike the L5-specific CYFIP2 reduction observed in adult Cyfip2+/- mice. Nevertheless, filamentous actin (F-actin) levels were increased only in L5 of Cyfip2 cKO mPFC possibly because of a compensatory increase in CYFIP1, the other member of CYFIP family, in L2/3 neurons. Abnormal dendritic spines on basal, but not on apical, dendrites were consistently observed in L5 neurons of Cyfip2 cKO mPFC. Meanwhile, neuronal excitability and activity were enhanced in both L2/3 and L5 neurons of Cyfip2 cKO mPFC, suggesting that CYFIP2 functions of regulating F-actin and excitability/activity may be mediated through independent mechanisms. Unexpectedly, adult Cyfip2 cKO mice did not display locomotor hyperactivity or reduced anxiety observed in Cyfip2+/- mice. Instead, both exhibited enhanced social dominance accessed by the tube test. Together, these results provide additional insights into the functions of CYFIP2 in the adult brain.

Differential cell-type-expression of CYFIP1 and CYFIP2 in the adult mouse hippocampus.

Recent molecular genetic studies have suggested that two members of the cytoplasmic FMR1-interacting protein (CYFIP) gene family, CYFIP1 and CYFIP2, are causally associated with several brain disorders. However, the clinical features of individuals with CYFIP1 and CYFIP2 variants are quite different. In addition, null mice for either Cyfip1 or Cyfip2 are lethal, indicating that these two genes cannot compensate for each other in vivo. Although these results strongly suggest that CYFIP1 and CYFIP2 have distinct functions in vivo, the detailed mechanisms underlying their differences remain enigmatic and unexplored, especially considering their high sequence homology. To address this, we analyzed a recently established mouse brain single-cell RNA sequencing (scRNAseq) database and found that Cyfip1 and Cyfip2 are dominantly expressed in non-neurons and neurons, respectively, in all tested brain regions. To validate these observations, we performed fluorescent immunohistochemistry in the adult mouse hippocampus with either a CYFIP1 or CYFIP2 antibody combined with antibodies for various cell-type-specific markers. Consistent with our analysis of the scRNAseq database, CYFIP1 signals were detected in both neurons and astrocytes, while CYFIP2 signals were mainly detected in neurons. These results suggest differential cell-type-expression of CYFIP1 and CYFIP2 in vivo, which provides novel insights into our understanding of the pathophysiology of and potential treatments for CYFIP-associated brain disorders.

Unexpected Compensatory Increase in Shank3 Transcripts in Shank3 Knock-Out Mice Having Partial Deletions of Exons.

Genetic variants of the SH3 and multiple ankyrin repeat domains 3 (SHANK3) gene, which encodes excitatory postsynaptic core scaffolds cause numerous brain disorders. Several lines of Shank3 knock-out (KO) mice with deletions of different Shank3 exons have previously been generated and characterized. The different Shank3 KO mouse lines have both common and line-specific phenotypes. Shank3 isoform diversity is considered a mechanism underlying phenotypic heterogeneity, and compensatory changes through regulation of Shank3 expression may contribute to this heterogeneity. However, whether such compensatory changes occur in Shank3 KO mouse lines has not been investigated in detail. Using previously reported RNA-sequencing analyses, we identified an unexpected increase in Shank3 transcripts in two different Shank3 mutant mouse lines (Shank3B and Shank3ΔC) having partial deletions of Shank3 exons. We validated an increase in Shank3 transcripts in the hippocampus, cortex, and striatum, but not in the cerebellum, of Shank3B heterozygous (HET) and KO mice, using qRT-PCR analyses. In particular, expression of the N-terminal exons 1-12, but not the more C-terminal exons 19-22, was observed to increase in Shank3B mice with deletion of exons 13-16. This suggests a selective compensatory activation of upstream Shank3 promoters. Furthermore, using domain-specific Shank3 antibodies, we confirmed that the increased Shank3 transcripts in Shank3B KO mice produced a small Shank3 isoform that was not detected in wild-type mice. Taken together, our results illustrate another layer of complexity in the regulation of Shank3 expression in the brain, which may also contribute to the phenotypic heterogeneity of different Shank3 KO mouse lines.