Fig 1.
Lethality and spontaneous recurrent seizures in adult Cyfip2+/R87C mice.
(A) Survival curves of Cyfip2+/R87C mice (on the C57BL/6N (N) background), Cyfip2+/− mice (on either the C57BL/6N or C57BL/6J (J) backgrounds), and their respective wild-type (WT) littermates, tracked from postnatal weeks 3 to 30 (log-rank test). (B) Behavioral seizure events during long-term (5-day) video recordings of Cyfip2+/R87C mice at postnatal weeks 14 (PWK 14) and 28 (PWK 28) (each group includes four mice, N1 to N4). Arrowheads indicate one-hour recording sessions with either one (black) or multiple (red) seizure events. Cyfip2+/R87C mouse N2 of PWK 28 died on day 5 of recording. The pie charts show the distribution of the number of seizure events per one-hour session at PWK 14 and PWK 28 (Fisher’s exact test). The line chart displays the number of seizure events during the 5-day recording for each genotype. Thin lines represent individual mice, while thick lines indicate the group averages. Note that no seizure events were observed in WT or Cyfip2+/− mice at PWK 28 (n = 4 mice per genotype). (C) Representative traces of EEG recordings from WT and Cyfip2+/R87C mice at PWK 20. Graphs show quantifications of the total number of seizures, tonic-clonic (T-C) seizures, and myoclonic jerks, as well as the duration (dur.) of T-C seizures and the amplitude (amp.) of myoclonic jerks (n = 3 mice per genotype, unpaired two-tailed Student t test). (D) Representative fluorescence immunohistochemistry images and quantification show a reduction in the CA1 region and dispersion of the dentate gyrus (DG) granule cell layer (GCL) in the hippocampus of Cyfip2+/R87C mice compared to WT mice at PWK 28 (n = 7 to 9 mice per genotype, two-way ANOVA with Šídák’s multiple comparisons test). Cyfip2+/− mice displayed a normal hippocampus, except for a reduction in DG height (H) compared to WT mice. T = thickness. *P < 0.05; **P < 0.01; ***P < 0.001. Data are represented as mean ± standard error of the mean. The data underlying this Figure can be found in S1 Data.
Fig 2.
Transcriptomic signatures in the cortex and hippocampus of Cyfip2+/R87C mice during seizure evolution.
(A) Volcano plots display downregulated (blue) and upregulated (red) differentially expressed genes (DEGs) in the cortex and hippocampus of Cyfip2+/R87C mice compared to WT mice at PWK 1, 7, and 14. Bar graphs present significant terms and their corresponding P values from the DAVID (Database for Annotation, Visualization, and Integrated Discovery) Gene Ontology (GO) analysis of the DEGs at PWK 14 (analyses for PWK 1 and 7 were not conducted due to the small number of DEGs). KEGG, Kyoto Encyclopedia of Genes and Genomes. (B) Volcano plots display DEGs in the cortex and hippocampus of Cyfip2+/− mice at PWK 1, 7, and 14. (C) Volcano plots display DEGs in the cortex and hippocampus of Cyfip2+/R87C mice at PWK 28. Bar graphs present significant terms and their corresponding P values from the DAVID GO analysis of the DEGs. (D) Gene Set Enrichment Analysis (GSEA) of the transcriptome from the cortex and hippocampus of Cyfip2+/R87C at PWK 1, 7, 14, and 28, focusing on neuron-, glia-, and other cell-type-specific gene sets. NES, normalized enrichment score, FDR, false discovery rate. (E) GSEA of the transcriptome from the cortex and hippocampus of Cyfip2+/R87C mice, focusing on biological process gene sets, along with clustering of the enriched gene sets using the Cytoscape EnrichmentMap.
Fig 3.
Molecular and structural synaptic remodeling in Cyfip2+/R87C mice during seizure evolution.
(A) Heat maps show qRT-PCR results for DEGs encoding synaptic proteins in the cortex and hippocampus of Cyfip2+/R87C mice compared to WT mice at various ages (n = 6 mice per genotype, two-way ANOVA with Šídák’s multiple comparisons test). Statistical significance of expression differences for each gene between PWK 14 and 28 within Cyfip2+/R87C mice is shown in the ‘PWK 14 vs. 28’ column. (B) Representative western blot images and heat maps display the expression levels of representative synaptic marker proteins in the cortical and hippocampal synaptosomes of Cyfip2+/R87C mice compared to WT mice at different ages (n = 6 to 8 mice per genotype, two-way ANOVA with Šídák’s multiple comparisons test). (C) Representative fluorescence immunohistochemistry images and quantification show changes in excitatory presynaptic (vGluT1) and postsynaptic (Homer1) markers in the hippocampal CA1 region of Cyfip2+/R87C mice compared to WT mice at PWK 14 and 28 (n = 7 to 9 mice per genotype, two-way ANOVA with Šídák’s multiple comparisons test). Images obtained using Imaris software for automated foci counting are also included. Scale bar, 2 μm. A.U. = arbitrary units. (D) Representative transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images and quantification of excitatory (Ex.) and inhibitory (Inh.) synapse numbers in the hippocampal CA1 region of WT and Cyfip2+/R87C mice at PWK 28 (n = 59 to 73 images from 3 mice per genotype, unpaired two-tailed Student t test). Presynaptic boutons form synaptic contacts on dendritic spines in WT mice, whereas in Cyfip2+/R87C mice, they contact dendritic shafts and cluster nearby. PSD, postsynaptic density. Scale bar, 0.5 μm. (E) 3D reconstruction of dendritic segments from CA1 pyramidal neurons in WT and Cyfip2+/R87C mice (left, without axons; right, with axons). The cumulative graph displays the distributions of PSD surface area in WT and Cyfip2+/R87C neurons (n = 46 to 77 PSDs per genotype, Kolmogorov–Smirnov test). *P < 0.05; **P < 0.01; ***P < 0.001. Data are represented as mean ± standard error of the mean. The data underlying this Figure can be found in S1 Data.
Fig 4.
Neuronal and glial changes in the hippocampus of Cyfip2+/R87C mice during seizure evolution.
(A) Representative fluorescence immunohistochemistry images illustrate age-dependent changes in the mean intensities of neuronal (NeuN) and astrocytic (GFAP) markers in the hippocampus of Cyfip2+/R87C mice compared to WT mice. DAPI was used for counterstaining nuclei. High-magnification images of the CA1 region at PWK 28 are shown. SO, stratum oriens, SP, stratum pyramidale, SR, stratum radiatum. (B) Representative fluorescence immunohistochemistry images show age-dependent changes in the mean intensities of oligodendrocytic (Olig2) and microglial (IBA1) markers in the hippocampus of Cyfip2+/R87C mice compared to WT mice. (C) Bar graphs display relative changes in mean intensities of neuronal and glial markers in the hippocampal CA1 region of Cyfip2+/R87C mice compared to age-matched WT mice (n = 7 to 9 mice per genotype, two-way ANOVA with Šídák’s multiple comparisons test). *P < 0.05; **P < 0.01; ***P < 0.001. Data are represented as mean ± standard error of the mean. The data underlying this Figure can be found in S1 Data.
Fig 5.
Morphological and functional changes of microglia in Cyfip2+/R87C mice.
(A) Representative 3D images of microglia in the hippocampal CA1 region of WT and Cyfip2+/R87C mice at PWK 14 and 28, processed using Imaris software. Graphs show quantifications from Sholl analysis as well as measurements of branch length, area, points, and diameter (n = 7 to 9 mice per genotype, two-way ANOVA with Šídák’s multiple comparisons test). (B) Representative fluorescence immunohistochemistry images and quantification show increased cleaved Caspase3 signals in microglia of the Cyfip2+/R87C hippocampus compared to the WT hippocampus at PWK 28, but not at PWK 14 (n = 7 to 9 mice per genotype, two-way ANOVA with Šídák’s multiple comparisons test). Arrows indicate microglia with enlarged images shown in the insets. SO, stratum oriens, SP, stratum pyramidale, SR, stratum radiatum. (C) Representative fluorescence immunohistochemistry images and quantification show increased CD68 signals in microglia of the Cyfip2+/R87C hippocampus compared to the WT hippocampus at PWK 14 (n = 7 to 8 mice per genotype, unpaired two-tailed Student t test). (D) Heat maps display qRT-PCR results for genes related to microglial activation in the cortex and hippocampus of Cyfip2+/R87C mice compared to WT mice at different ages (n = 6 mice per genotype, two-way ANOVA with Šídák’s multiple comparisons test). Statistical significance of expression differences for each gene between PWK 14 and 28 within Cyfip2+/R87C mice is shown in the ‘PWK 14 vs. 28’ column. Note that Il-6 was not detected in the cortex at PWK 1. (E) Representative 3D images illustrate microglia (IBA1), lysosome (Lamp1) within the microglia, and excitatory synaptic protein (Homer1) within microglial lysosome (i.e., engulfed Homer1, indicated by white arrows in merged images) in the hippocampal CA1 region of WT and Cyfip2+/R87C mice at PWK 14, analyzed using Imaris software. Graphs show quantifications of microglial lysosome and engulfed Homer1 in WT and Cyfip2+/R87C mice (n = 7 mice per genotype, unpaired two-tailed Student t test). *P < 0.05; **P < 0.01; ***P < 0.001. Data are represented as mean ± standard error of the mean. The data underlying this Figure can be found in S1 Data.
Fig 6.
Changes in morphology and lipid droplets of astrocytes in Cyfip2+/R87C mice.
(A) Representative 3D images of astrocytes in the hippocampal CA1 region of WT and Cyfip2+/R87C mice at PWK 14 and 28, processed using Imaris software. Graphs show quantifications from Sholl analysis as well as measurements of branch length, area, points, and diameter (n = 7 to 8 mice per genotype, two-way ANOVA with Šídák’s multiple comparisons test). (B) Serial electron microscopic images of lysosomes containing lipid droplets (LDs) in the hippocampal astrocytes of WT and Cyfip2+/R87C mice at PWK 28. 3D-rendered images of these lysosomes are also shown. (C) Correlative light and electron microscopy (CLEM) image of the hippocampal astrocyte in Cyfip2+/R87C mouse at PWK 28 shows BODIPY (light microscopy, LM), a marker for neutral lipids, within electron-dense lysosome observed by electron microscopy (EM). (D) Representative 3D images illustrate astrocytes (GFAP), lysosome (Lamp1) within the astrocytes, and LDs (BODIPY) within astrocytic lysosome (indicated by white arrows in merged images) in the hippocampus of WT and Cyfip2+/R87C mice at PWK 28, analyzed using Imaris software. Graphs show quantifications of the number and volume of LDs within astrocytic lysosomes in WT and Cyfip2+/R87C mice (n = 3 to 8 mice per genotype, unpaired two-tailed Student t test). (E) Serial electron microscopic images of a lipid droplet with crystals in the hippocampal astrocyte of Cyfip2+/R87C mouse at PWK 28. A 3D-rendered image of this lipid droplet is also presented. (F) CLEM image of the hippocampal astrocyte in Cyfip2+/R87C mouse at PWK 28 shows reflection signals from LM that match the crystals observed by EM. The graph displays the number of crystal spots, as measured by reflection signals from LM, in the hippocampus of WT and Cyfip2+/R87C mice at PWK 28 (n = 5 to 12 mice per genotype, unpaired two-tailed Student t test). *P < 0.05; **P < 0.01; ***P < 0.001. Data are represented as mean ± standard error of the mean. The data underlying this Figure can be found in S1 Data.
Fig 7.
Single-nucleus transcriptomic analysis on the hippocampus of Cyfip2+/R87C mice.
(A) UMAP visualization of 12 hippocampal cell types from WT and Cyfip2+/R87C mice at PWK 28. (B) A dot plot illustrating the expression of cell-specific genes used to identify the 12 cell types, with dot size representing the percentage of cells expressing the gene and color indicating the average (Avg.) expression level. (C) Violin plot showing the expression levels of Apod and Ptgds in different cell types, grouped by genotype. (D) Subclustering results of CA1 neurons. The left panel presents a UMAP visualization of CA1 neuronal subtypes, while the middle panel features a dot plot highlighting the top DEGs for each cluster. The right panel shows the results of MiloR analysis, illustrating CA1 abundance differences, with genotypes color-coded as WT (blue) and Cyfip2+/R87C (red). (E) Heatmap displaying Over-Representation Analysis (ORA) results for regulons regulated by transcription factors (TFs) with higher activity in CA1-1 compared to CA1-0. Color intensity represents similarity, and each label corresponds to the ancestor term of the respective cluster. (F) Subclustering results of astrocytes. (G) The left panel shows a UMAP visualization of abundance analysis results, illustrating differences in astrocyte abundance between WT (blue) and Cyfip2+/R87C (red) mice. The right panel displays a volcano plot illustrating the results of the differential expression (DE) test between Astrocyte-4 and other subtypes. (H) Heatmap illustrating the ORA results for regulons governed by TFs with higher activity in Astrocyte-4 compared to Astrocyte-1. (I) Heatmap showing GSEA results for Astrocyte-4 compared to Astrocyte-1. (J) Ligand-receptor (LR) interactions between subtypes of neurons and astrocytes. Interactions were inferred from the expression profiles of ligands and receptors across different cell populations. LR interactions specific to Cyfip2+/R87C neuron-astrocyte pairs (CA1-1 to Astrocyte-4 and Astrocyte-4 to CA1-1) are highlighted in red. (K) Diagrams depicting bidirectional interactions between CA1-1 and Astrocyte-4, illustrating the potential influence of ligands on target cell gene expression (blue, downregulated; red, upregulated).
Fig 8.
Proteomic and lipidomic changes in the hippocampus of Cyfip2+/R87C mice.
(A) Principal component analysis (PCA) score plots show distinct separations in the proteome between WT and Cyfip2+/R87C mice at PWK 14 and 28. (B) Heatmaps display 3,102 and 3,748 differentially expressed proteins (DEPs) in the hippocampus of Cyfip2+/R87C mice at PWK 14 and 28, respectively. (C) Graphs depict the abundance of Apod and Ptgds proteins in WT and Cyfip2+/R87C mice (n = 8 mice per genotype, Welch’s t test). KI, knock-in. (D) Heatmap illustrates the separation of four distinct protein groups in the proteome of WT and Cyfip2+/R87C mice, categorized based on age-dependent relative abundance differences between the genotypes. (E) GO analysis of proteins in groups B and D (left panel). Protein–protein interaction (PPI) networks for group D proteins, highlighting their associated metabolic processes (right panel). (F) Representative electron microscopy images show astrocytic mitochondria (indicated by red arrows) in the hippocampus of WT and Cyfip2+/R87C mice at PWK 28. (G) PCA score plots display clear separations in the lipidome between WT and Cyfip2+/R87C mice at PWK 14 and 28. (H) A pie chart illustrates the distribution of identified lipid classes in the hippocampus. (I) Pie charts depict the distribution of identified lipid classes across the six groups classified based on the age-dependent relative expression levels in Cyfip2+/R87C mice compared to WT mice. Bar graphs present Z-scores for the four combinations (two ages and two genotypes) across the six groups (n = 9 to 14 mice per genotype, Mann–Whitney test). *P < 0.05; **P < 0.01; ***P < 0.001. Data are represented as mean ± standard error of the mean. The data underlying this Figure can be found in S1 Data.
Fig 9.
Summary of the seizure evolution in Cyfip2+/R87C mice.
Seizure phenotypes in Cyfip2+/R87C mice begin with infantile spasms during the neonatal stage (PWK 1), followed by a seizure-free latent period during the juvenile and young adult stages. Around PWK 14, Cyfip2+/R87C mice start to exhibit spontaneous recurrent seizures, which worsen with age and are associated with premature death. This seizure evolution is accompanied by various temporal cellular and molecular changes in the brain, including alterations in neurons, glial cells, and their lipid and metabolic profiles, which may interact to contribute to the seizure phenotype.