Cntnap2-dependent molecular networks in autism spectrum disorder revealed through an integrative multi-omics analysis

Abstract

Autism spectrum disorder (ASD) is a major neurodevelopmental disorder in which patients present with core symptoms of social communication impairment, restricted interest, and repetitive behaviors. Although various studies have been performed to identify ASD-related mechanisms, ASD pathology is still poorly understood. CNTNAP2 genetic variants have been found that represent ASD genetic risk factors, and disruption of Cntnap2 expression has been associated with ASD phenotypes in mice. In this study, we performed an integrative multi-omics analysis by combining quantitative proteometabolomic data obtained with Cntnap2 knockout (KO) mice with multi-omics data obtained from ASD patients and forebrain organoids to elucidate Cntnap2-dependent molecular networks in ASD. To this end, a mass spectrometry-based proteometabolomic analysis of the medial prefrontal cortex in Cntnap2 KO mice led to the identification of Cntnap2-associated molecular features, and these features were assessed in combination with multi-omics data obtained on the prefrontal cortex in ASD patients to identify bona fide ASD cellular processes. Furthermore, a reanalysis of single-cell RNA sequencing data obtained from forebrain organoids derived from patients with CNTNAP2-associated ASD revealed that the aforementioned identified ASD processes were mainly linked to excitatory neurons. On the basis of these data, we constructed Cntnap2-associated ASD network models showing mitochondrial dysfunction, axonal impairment, and synaptic activity. Our results may shed light on the Cntnap2-dependent molecular networks in ASD.

Fig. 1. Impaired social preference in Cntnap2 KO mice.

a Schematic diagram used in the three-chamber social behavior tests. M: target conspecific; O: inanimate object. b Bar graph showing the percentage of the time spent toward a target conspecific or an inanimate object in control (n = 15) and Cntnap2 KO (n = 16) mice. Two-way ANOVA p = 0.0045; Sidak’s multiple comparison test, Control: mice vs object ****p < 0.0001, Cntnap2 KO: mice vs object p = 0.0634. c Bar graph showing preference index with significant differences between Cntnap2 KO and control mice. Two-tailed Welch’s t-test *p = 0.0441.

Fig. 2. Proteomic alterations associated with Cntnap2 KO mPFC in mice.

a Workflow of quantitative proteomic analysis. b Heatmap showing the expressional difference of DEPeptides between the Cntnap2 KO and control groups. c, d The relative proportion of DEPs according to their GOBP involvement at GOBP c level 1 and d GOBP levels 2-5. e Cellular processes are enriched with upregulated and downregulated proteins.

Fig. 3. Metabolomic alterations associated with Cntnap2 KO mPFC in mice.

a Workflow of targeted quantitative metabolomic analysis. b PLS-DA plot showing the discrimination between Cntnap2 KO from the control group. c The bar graph shows the number of measured metabolites in each metabolite subclass with unchanged metabolites and DEMs in each stacked bar, respectively. (d) Box plot showing DEM concentrations between Cntnap2 KO and control groups.

Fig. 4. Molecular overlapping between Cntnap2 KO mice and ASD patients.

a Venn diagram showing the number of comparable genes in our Cntnap2 KO mPFC proteome and the public human PFC transcriptome. b Pivot table showing the number of genes with corresponding expression patterns of our proteome and PFC transcriptome of ASD patients. c Venn diagram showing the number of comparable metabolites in our Cntnap2 KO mPFC metabolome and the public human PFC lipidome/metabolome. d Pivot table showing the number of metabolites with corresponding expression patterns of our metabolome and ASD patients PFC lipidome/metabolome. e Heatmaps shows the expression differences of genes or metabolites in mouse and human ASD subjects compared to their corresponding controls. The genes reported as ASD-linked genes in SFARI are labeled in bold. f Radar plot shows cellular processes enriched with upregulated and downregulated genes in mouse and human ASD subjects.

Fig. 5. CNTNAP2-dependent cell types and the specific processes in which they are involved in CNTNAP2-deficient ASD patient-derived brain organoids.

a UMAP visualization of the 5 representative cell types identified in the forebrain organoid DPal cells. See color legend for the cell type assignment. b Heatmap showing the significance of cellular processes enriched with DEPs, DEGs, and cell-type-specific DEGs identified in the mouse proteome results, ASD patient integration results, and ASD organoid results. c Expression of CNTNAP2 in ASD and healthy organoid DPal cells. d Dot plot showing cell-type-specific CNTNAP2 expression. The dot size and color represent the proportion of CNTNAP2-expressing cells and average CNTNAP2 expression per cell-type cluster, respectively. e Functional protein association network of genes shown in both Cntnap2 KO DEP and Ex-specific DEG. The node color represents the expressional difference between ASD and healthy organoids in Ex cells. The edge thickness represents the STRING interaction score.

Fig. 6. Cntnap2-associated ASD molecular network models.

Molecular network models describing alterations in a myelin sheath and mitochondrial network, b synapses, and c neuron projections in the CNTNAP2-associated ASD PFC. Node shapes represent types of molecules. The molecules showing a significant expressional difference in mouse and human ASD subjects are displayed as larger nodes. The center and boundary colors of a node represent the expressional difference in the ASD mouse and ASD organoid Ex cells compared to their corresponding controls, respectively. Line types indicate types of regulation. Thin gray lines represent the curated protein-protein interaction.