Capicua (CIC) has been implicated in pathogenesis of spinocerebellar ataxia type 1 and cancer in mammals; however, the in vivo physiological functions of CIC remain largely unknown. Here we show that Cic hypomorphic (Cic-L(-/-)) mice have impaired bile acid (BA) homeostasis associated with induction of proinflammatory cytokines. We discovered that several drug metabolism and BA transporter genes were down-regulated in Cic-L(-/-) liver, and that BA was increased in the liver and serum whereas bile was decreased within the gallbladder of Cic-L(-/-) mice. We also found that levels of proinflammatory cytokine genes were up-regulated in Cic-L(-/-) liver. Consistent with this finding, levels of hepatic transcriptional regulators, such as hepatic nuclear factor 1 alpha (HNF1α), CCAAT/enhancer-binding protein beta (C/EBPβ), forkhead box protein A2 (FOXA2), and retinoid X receptor alpha (RXRα), were markedly decreased in Cic-L(-/-) mice. Moreover, induction of tumor necrosis factor alpha (Tnfα) expression and decrease in the levels of FOXA2, C/EBPβ, and RXRα were found in Cic-L(-/-) liver before BA was accumulated, suggesting that inflammation might be the cause for the cholestasis in Cic-L(-/-) mice. Our findings indicate that CIC is a critical regulator of BA homeostasis, and that its dysfunction might be associated with chronic liver disease and metabolic disorders.

Spinocerebellar ataxia type 1 (SCA1) is a paradigmatic neurodegenerative proteinopathy, in which a mutant protein (in this case, ATAXIN1) accumulates in neurons and exerts toxicity; in SCA1, this process causes progressive deterioration of motor coordination. Seeking to understand how post-translational modification of ATAXIN1 levels influences disease, we discovered that the RNA-binding protein PUMILIO1 (PUM1) not only directly regulates ATAXIN1 but also plays an unexpectedly important role in neuronal function. Loss of Pum1 caused progressive motor dysfunction and SCA1-like neurodegeneration with motor impairment, primarily by increasing Ataxin1 levels. Breeding Pum1(+/-) mice to SCA1 mice (Atxn1(154Q/+)) exacerbated disease progression, whereas breeding them to Atxn1(+/-) mice normalized Ataxin1 levels and largely rescued the Pum1(+/-) phenotype. Thus, both increased wild-type ATAXIN1 levels and PUM1 haploinsufficiency could contribute to human neurodegeneration. These results demonstrate the importance of studying post-transcriptional regulation of disease-driving proteins to reveal factors underlying neurodegenerative disease.

We describe the landscape of somatic genomic alterations of 66 chromophobe renal cell carcinomas (ChRCCs) on the basis of multidimensional and comprehensive characterization, including mtDNA and whole-genome sequencing. The result is consistent that ChRCC originates from the distal nephron compared with other kidney cancers with more proximal origins. Combined mtDNA and gene expression analysis implicates changes in mitochondrial function as a component of the disease biology, while suggesting alternative roles for mtDNA mutations in cancers relying on oxidative phosphorylation. Genomic rearrangements lead to recurrent structural breakpoints within TERT promoter region, which correlates with highly elevated TERT expression and manifestation of kataegis, representing a mechanism of TERT upregulation in cancer distinct from previously observed amplifications and point mutations.

Although expansion of CAG repeats in ATAXIN1 (ATXN1) causes Spinocerebellar ataxia type 1, the functions of ATXN1 and ATAXIN1-Like (ATXN1L) remain poorly understood. To investigate the function of these proteins, we generated and characterized Atxn1L(-/-) and Atxn1(-/-); Atxn1L(-/-) mice. Atxn1L(-/-) mice have hydrocephalus, omphalocele, and lung alveolarization defects. These phenotypes are more penetrant and severe in Atxn1(-/-); Atxn1L(-/-) mice, suggesting that ATXN1 and ATXN1L are functionally redundant. Upon pursuing the molecular mechanism, we discovered that several Matrix metalloproteinase (Mmp) genes are overexpressed and that the transcriptional repressor Capicua (CIC) is destabilized in Atxn1L(-/-) lungs. Consistent with this, Cic deficiency causes lung alveolarization defect. Loss of either ATXN1L or CIC derepresses Etv4, an activator for Mmp genes, thereby mediating MMP9 overexpression. These findings demonstrate a critical role of ATXN1/ATXN1L-CIC complexes in extracellular matrix (ECM) remodeling during development and their potential roles in pathogenesis of disorders affecting ECM remodeling.

Variants of the SHANK3 gene, which encodes a core scaffold protein of the postsynaptic density of excitatory synapses, have been causally associated with numerous brain disorders. Shank3 proteins directly bind zinc ions through their C-terminal sterile α motif domain, which enhances the multimerization and synaptic localization of Shank3, to regulate excitatory synaptic strength. However, no studies have explored whether zinc affects the protein interactions of Shank3, which might contribute to the synaptic changes observed after zinc application. To examine this, we first purified Shank3 protein complexes from mouse brain synaptosomal lysates that were incubated with different concentrations of ZnCl2, and analyzed them with mass spectrometry. We used strict criteria to identify 71 proteins that specifically interacted with Shank3 when extra ZnCl2 was added to the lysate. To characterize the zinc-induced Shank3 interactome, we performed various bioinformatic analyses that revealed significant associations of the interactome with subcellular compartments, including mitochondria, and brain disorders, such as bipolar disorder and schizophrenia. Together, our results showing that zinc affected the Shank3 protein interactions of in vitro mouse synaptosomes provided an additional link between zinc and core synaptic proteins that have been implicated in multiple brain disorders.

Mania causes symptoms of hyperactivity, impulsivity, elevated mood, reduced anxiety and decreased need for sleep, which suggests that the dysfunction of the striatum, a critical component of the brain motor and reward system, can be causally associated with mania. However, detailed molecular pathophysiology underlying the striatal dysfunction in mania remains largely unknown. In this study, we aimed to identify the molecular pathways showing alterations in the striatum of SH3 and multiple ankyrin repeat domains 3 (Shank3)-overexpressing transgenic (TG) mice that display manic-like behaviors. The results of transcriptome analysis suggested that mammalian target of rapamycin complex 1 (mTORC1) signaling may be the primary molecular signature altered in the Shank3 TG striatum. Indeed, we found that striatal mTORC1 activity, as measured by mTOR S2448 phosphorylation, was significantly decreased in the Shank3 TG mice compared to wild-type (WT) mice. To elucidate the potential underlying mechanism, we re-analyzed previously reported protein interactomes, and detected a high connectivity between Shank3 and several upstream regulators of mTORC1, such as tuberous sclerosis 1 (TSC1), TSC2 and Ras homolog enriched in striatum (Rhes), via 94 common interactors that we denominated "Shank3-mTORC1 interactome". We noticed that, among the 94 common interactors, 11 proteins were related to actin filaments, the level of which was increased in the dorsal striatum of Shank3 TG mice. Furthermore, we could co-immunoprecipitate Shank3, Rhes and Wiskott-Aldrich syndrome protein family verprolin-homologous protein 1 (WAVE1) proteins from the striatal lysate of Shank3 TG mice. By comparing with the gene sets of psychiatric disorders, we also observed that the 94 proteins of Shank3-mTORC1 interactome were significantly associated with bipolar disorder (BD). Altogether, our results suggest a protein interaction-mediated connectivity between Shank3 and certain upstream regulators of mTORC1 that might contribute to the abnormal striatal mTORC1 activity and to the manic-like behaviors of Shank3 TG mice.

Recent molecular genetic studies have identified 100s of risk genes for various neurodevelopmental and neuropsychiatric disorders. As the number of risk genes increases, it is becoming clear that different mutations of a single gene could cause different types of disorders. One of the best examples of such a gene is SHANK3, which encodes a core scaffold protein of the neuronal excitatory post-synapse. Deletions, duplications, and point mutations of SHANK3 are associated with autism spectrum disorders, intellectual disability, schizophrenia, bipolar disorder, and attention deficit hyperactivity disorder. Nevertheless, how the different mutations of SHANK3 can lead to such phenotypic diversity remains largely unknown. In this study, we investigated whether Shank3 could form protein complexes in a brain region-specific manner, which might contribute to the heterogeneity of neuronal pathophysiology caused by SHANK3 mutations. To test this, we generated a medial prefrontal cortex (mPFC) Shank3 in vivo interactome consisting of 211 proteins, and compared this protein list with a Shank3 interactome previously generated from mixed hippocampal and striatal (HP+STR) tissues. Unexpectedly, we found that only 47 proteins (about 20%) were common between the two interactomes, while 164 and 208 proteins were specifically identified in the mPFC and HP+STR interactomes, respectively. Each of the mPFC- and HP+STR-specific Shank3 interactomes represents a highly interconnected network. Upon comparing the brain region-enriched proteomes, we found that the large difference between the mPFC and HP+STR Shank3 interactomes could not be explained by differential protein expression profiles among the brain regions. Importantly, bioinformatic pathway analysis revealed that the representative biological functions of the mPFC- and HP+STR-specific Shank3 interactomes were different, suggesting that these interactors could mediate the brain region-specific functions of Shank3. Meanwhile, the same analysis on the common Shank3 interactors, including Homer and GKAP/SAPAP proteins, suggested that they could mainly function as scaffolding proteins at the post-synaptic density. Lastly, we found that the mPFC- and HP+STR-specific Shank3 interactomes contained a significant number of proteins associated with neurodevelopmental and neuropsychiatric disorders. These results suggest that Shank3 can form protein complexes in a brain region-specific manner, which might contribute to the pathophysiological and phenotypic diversity of disorders related to SHANK3 mutations.

We evaluated the association between prostate cancer non-coding RNA 1 (PRNCR1) polymorphisms and the risk of developing gastric cancer (GC) and GC subgroups in Korea. A case-control study was conducted with 437 GC patients and 357 healthy controls using a TaqMan genotyping assay. A chi-squared test, binary logistic regression, and genetic models were used to explore the association between five PRNCR1 polymorphisms and GC risk. After adjusting for gender and age, overall analyses using the recessive model indicated that the rs13252298 GG genotype was significantly associated with increased risk of intestinal-type gastric cancer (IGC). In the stratification analyses, the recessive model indicated that the rs1016343 TT genotype was significantly associated with decreased GC risk in individuals aged <60 years showing lymph node metastasis (LNM)-negative results. The rs13252298 GG genotype in the recessive model showed increased GC risk in subjects aged ≥60 years showing LNM-positive results and those aged ≥60 years in tumor stage III. In the dominant model, the rs16901946 combined genotype (AG/GG) was significantly associated with increased GC risk in subjects aged <60 years with tumor stage III. In the recessive model, the rs16901946 GG genotype was associated with decreased risk of GC and IGC in males aged ≥60 years. Thus, genetic variations in PRNCR1 may contribute to susceptibility to GC.

Mutations in SHANK3 and large duplications of the region spanning SHANK3 both cause a spectrum of neuropsychiatric disorders, indicating that proper SHANK3 dosage is critical for normal brain function. However, SHANK3 overexpression per se has not been established as a cause of human disorders because 22q13 duplications involve several genes. Here we report that Shank3 transgenic mice modelling a human SHANK3 duplication exhibit manic-like behaviour and seizures consistent with synaptic excitatory/inhibitory imbalance. We also identified two patients with hyperkinetic disorders carrying the smallest SHANK3-spanning duplications reported so far. These findings indicate that SHANK3 overexpression causes a hyperkinetic neuropsychiatric disorder. To probe the mechanism underlying the phenotype, we generated a Shank3 in vivo interactome and found that Shank3 directly interacts with the Arp2/3 complex to increase F-actin levels in Shank3 transgenic mice. The mood-stabilizing drug valproate, but not lithium, rescues the manic-like behaviour of Shank3 transgenic mice raising the possibility that this hyperkinetic disorder has a unique pharmacogenetic profile.

ΔNp63 is required for both the proliferation and differentiation of keratinocytes, but its role in the differentiation of these cells is poorly understood. The corresponding gene, TP63, harbors the MIR944 sequence within its intron. However, the mechanism of biogenesis and the function of miR-944 are unknown. We found that miR-944 is highly expressed in keratinocytes, in a manner that is concordant with that of ΔNp63 mRNA, but the regulation of miR-944 expression under various conditions did not correspond with that of ΔNp63. Bioinformatics analysis and functional studies demonstrated that MIR944 has its own promoter. We demonstrate here that MIR944 is a target of ΔNp63. Promoter analysis revealed that the activity of the MIR944 promoter was markedly enhanced by the binding of ΔNp63, which was maintained by the supportive action of AP-2 during keratinocyte differentiation. Our results indicated that miR-944 biogenesis is dependent on ΔNp63 protein, even though it is generated from ΔNp63 mRNA-independent transcripts. We also demonstrated that miR-944 induces keratin 1 and keratin 10 expression by inhibiting ERK signaling and upregulating p53 expression. Our findings suggested that miR-944, as an intronic miRNA and a direct target of ΔNp63, contributes to the function of ΔNp63 in the induction of epidermal differentiation.

The extent and pattern of linkage disequilibrium (LD) in the human genome provide important information for disease gene mapping. Previous studies have shown that LDs vary depending on chromosomal regions and populations. As the Asian samples of the International HapMap Project consisted of Japanese and Chinese populations, it was of interest whether we could use the HapMap data as a reference to carry out association studies of common complex diseases in a closely related population, such as Koreans. We have compared the LD and recombination patterns defined by single-nucleotide polymorphisms (SNPs) in ENCODE region ENm010, chromosome 7p15.2, in Korean, Japanese, and Chinese samples and further tested the robustness of tagSNPs among the Asian samples. We genotyped 792 SNPs in 500 kb (chromosome 7: 26699793-27199792, NCBI build 34) from 90 unrelated Koreans by fluorescence polarization detection and compared the data with Asian data from the HapMap project. Despite some differences in the position of high LD region boundaries, the overall patterns of LD were remarkably similar across the three samples, reflecting strong genetic affinities among them. Furthermore, the haplotype tag SNP transferability across the three samples was greater than 90%. Our results support the initial suggestion that the populations genotyped in the HapMap project might serve as reference populations for the selection of tagSNPs in association studies.

Cytoplasmic FMR1-interacting protein 2 (CYFIP2) is a key component of the WAVE regulatory complex (WRC) which regulates actin polymerization and branching in diverse cellular compartments. Recent whole exome sequencing studies identified de novo hotspot variants in CYFIP2 from patients with early-onset epileptic encephalopathy and microcephaly, suggesting that CYFIP2 may have some functions in embryonic brain development. Although perinatal lethality of Cyfip2-null (Cyfip2 -/-) mice was reported, the exact developmental time point and cause of lethality, and whether Cyfip2 -/- embryonic mice have brain abnormalities remain unknown. We found that endogenous Cyfip2 is mainly expressed in the brain, spinal cord, and thymus of mice at late embryonic stages. Cyfip2 -/- embryos did not show lethality at embryonic day 18.5 (E18.5), but their body size was smaller than that of wild-type (WT) or Cyfip2 +/- littermates. Meanwhile, at postnatal day 0, all identified Cyfip2 -/- mice were found dead, suggesting early postnatal lethality of the mice. Nevertheless, the brain size and cortical cytoarchitecture were comparable among WT, Cyfip2 +/-, and Cyfip2 -/- mice at E18.5. Using RNA-sequencing analyses, we identified 98 and 72 differentially expressed genes (DEGs) from the E18.5 cortex of Cyfip2 +/- and Cyfip2 -/- mice, respectively. Further bioinformatic analyses suggested that extracellular matrix (ECM)-related gene expression changes in Cyfip2 -/- embryonic cortex. Together, our results suggest that CYFIP2 is critical for embryonic body growth and for early postnatal survival, and that loss of its expression leads to ECM-related gene expression changes in the embryonic cortex without severe gross morphological defects.

This study aimed to extend the retention of flavor in coffee-containing milk beverage by microencapsulation. The core material was caramel flavor, and the primary and secondary coating materials were medium-chain triglyceride and maltodextrin, respectively. Polyglycerol polyricinoleate was used as the primary emulsifier, and the secondary emulsifier was polyoxyethylene sorbitan monolaurate. Response surface methodology was employed to determine optimum microencapsulation conditions, and headspace solid-phase microextraction was used to detect the caramel flavor during storage. The microencapsulation yield of the caramel flavor increased as the ratio of primary to secondary coating material increased. The optimum ratio of core to primary coating material for the water-in-oil (W/O) phase was 1:9, and that of the W/O phase to the secondary coating material was also 1:9. Microencapsulation yield was observed to be approximately 93.43%. In case of in vitro release behavior, the release rate of the capsules in the simulated gastric environment was feeble; however, the release rate in the simulated intestinal environment rapidly increased within 30 min, and nearly 70% of the core material was released within 120 min. The caramel flavor-supplemented beverage sample exhibited an exponential degradation in its flavor components. However, microcapsules containing flavor samples showed sustained flavor release compared to caramel flavor-filled samples under higher storage temperatures. In conclusion, the addition of coffee flavor microcapsules to coffee-containing milk beverages effectively extended the retention of the coffee flavor during the storage period.

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.

Myelin transcription factor 1 like (Myt1l), a zinc-finger transcription factor, promotes neuronal differentiation and is implicated in autism spectrum disorder (ASD) and intellectual disability. However, it remains unclear whether Myt1l promotes neuronal differentiation in vivo and its deficiency in mice leads to disease-related phenotypes. Here, we report that Myt1l-heterozygous mutant (Myt1l-HT) mice display postnatal age-differential ASD-related phenotypes: newborn Myt1l-HT mice, with strong Myt1l expression, show ASD-like transcriptomic changes involving decreased synaptic gene expression and prefrontal excitatory synaptic transmission and altered righting reflex. Juvenile Myt1l-HT mice, with markedly decreased Myt1l expression, display reverse ASD-like transcriptomes, increased prefrontal excitatory transmission, and largely normal behaviors. Adult Myt1l-HT mice show ASD-like transcriptomes involving astrocytic and microglial gene upregulation, increased prefrontal inhibitory transmission, and behavioral deficits. Therefore, Myt1l haploinsufficiency leads to ASD-related phenotypes in newborn mice, which are temporarily normalized in juveniles but re-appear in adults, pointing to continuing phenotypic changes long after a marked decrease of Myt1l expression in juveniles.

Hair cells are sensory receptors for the auditory and vestibular system in vertebrates. The transcription factor Atoh1 is both necessary and sufficient for the differentiation of hair cells, and is strongly upregulated during hair-cell regeneration in nonmammalian vertebrates. To identify genes involved in hair cell development and function, we performed RNA-seq profiling of purified Atoh1-expressing hair cells from the neonatal mouse cochlea. We identified >600 enriched transcripts in cochlear hair cells, of which 90% have not been previously shown to be expressed in hair cells. We identified 233 of these hair cell genes as candidates to be directly regulated by Atoh1 based on the presence of Atoh1 binding sites in their regulatory regions and by analyzing Atoh1 ChIP-seq datasets from the cerebellum and small intestine. We confirmed 10 of these genes as being direct Atoh1 targets in the cochlea by ChIP-PCR. The identification of candidate Atoh1 target genes is a first step in identifying gene regulatory networks for hair-cell development and may inform future studies on the potential role of Atoh1 in mammalian hair cell regeneration.

SLC6A20A is a proline and glycine transporter known to regulate glycine homeostasis and NMDA receptor (NMDAR) function in the brain. A previous study found increases in ambient glycine levels and NMDA receptor-mediated synaptic transmission in the brains of Slc6a20a-haploinsufficient mice, but it remained unknown whether Slc6a20a deficiency leads to disease-related behavioral deficits in mice. Here, we report that Slc6a20a heterozygous and homozygous mutant mice display differential behavioral phenotypes in locomotor, repetitive behavioral, and spatial and fear memory domains. In addition, these mice show differential transcriptomic changes in synapse, ribosome, mitochondria, autism, epilepsy, and neuron-related genes. These results suggest that heterozygous and homozygous Slc6a20a deletions in mice lead to differential changes in behaviors and transcriptomes.

Shank2 is an excitatory postsynaptic scaffolding protein strongly implicated in autism spectrum disorders (ASDs). Shank2-mutant mice with a homozygous deletion of exons 6 and 7 (Shank2-KO mice) show decreased NMDA receptor (NMDAR) function and autistic-like behaviors at juvenile [∼postnatal day (P21)] and adult (>P56) stages that are rescued by NMDAR activation. However, at ∼P14, these mice show the opposite change - increased NMDAR function; moreover, suppression of NMDAR activity with early, chronic memantine treatment during P7-21 prevents NMDAR hypofunction and autistic-like behaviors at later (∼P21 and >P56) stages. To better understand the mechanisms underlying this rescue, we performed RNA-Seq gene-set enrichment analysis of forebrain transcriptomes from wild-type (WT) and Shank2-KO juvenile (P25) mice treated early and chronically (P7-21) with vehicle or memantine. Vehicle-treated Shank2-KO mice showed upregulation of synapse-related genes and downregulation of ribosome- and mitochondria-related genes compared with vehicle-treated WT mice. They also showed a transcriptomic pattern largely opposite that observed in ASD (reverse-ASD pattern), based on ASD-related/risk genes and cell-type-specific genes. In memantine-treated Shank2-KO mice, chromatin-related genes were upregulated; mitochondria, extracellular matrix (ECM), and actin-related genes were downregulated; and the reverse-ASD pattern was weakened compared with that in vehicle-treated Shank2-KO mice. In WT mice, memantine treatment, which does not alter NMDAR function, upregulated synaptic genes and downregulated ECM genes; memantine-treated WT mice also exhibited a reverse-ASD pattern. Therefore, early chronic treatment of Shank2-KO mice with memantine alters expression of chromatin, mitochondria, ECM, actin, and ASD-related genes.

Early exposures to anesthetics can cause long-lasting changes in excitatory/inhibitory synaptic transmission (E/I imbalance), an important mechanism for neurodevelopmental disorders. Since E/I imbalance is also involved with addiction, we further investigated possible changes in addiction-related behaviors after multiple ketamine anesthesia in late postnatal mice. Postnatal day (PND) 16 mice received multiple ketamine anesthesia (35 mg kg-1, 5 days), and behavioral changes were evaluated at PND28 and PND56. Although mice exposed to early anesthesia displayed normal behavioral sensitization, we found significant increases in conditioned place preference to both low-dose ketamine (20 mg kg-1) and nicotine (0.5 mg kg-1). By performing transcriptome analysis and whole-cell recordings in the hippocampus, a brain region involved with CPP, we also discovered enhanced neuronal excitability and E/I imbalance in CA1 pyramidal neurons. Interestingly, these changes were not found in female mice. Our results suggest that repeated ketamine anesthesia during neurodevelopment may influence drug reward behavior later in life.

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.