Deregulated mRNA and microRNA Expression Patterns in the Prefrontal Cortex of the BTBR Mouse Model of Autism

Abstract

Autism spectrum disorder (ASD) is a neurodevelopmental condition caused by both genetic and environmental factors. Since no single gene variant accounts for more than 1% of the cases, the converging actions of ASD-related genes and other factors, including microRNAs (miRNAs), may contribute to ASD pathogenesis. To date, few studies have simultaneously investigated the mRNA and miRNA profiles in an ASD-relevant model. The BTBR mouse strain displays a range of behaviors with ASD-like features but little is known about the protein-coding and noncoding gene expression landscape that may underlie the ASD-like phenotype. Here we performed parallel mRNA and miRNA profiling using the prefrontal cortex (PFC) of BTBR and C57BL/6 J (B6) mice. This identified 1063 differentially expressed genes and 48 differentially expressed miRNAs. Integration of mRNA and miRNA data identified a strong inverse relationship between upregulated (DEGs) and downregulated miRNAs, and vice versa. Pathway analysis, taking account of the inverse relationship between differentially expressed miRNAs and their target mRNAs highlighted significant shared enrichment in immune signaling, myelination, and neurodevelopmental processes. Notably, miRNA changes were predicted to affect synapse-related functions but we did not find enrichment of protein-coding genes linked to cellular components or biological processes related to synapses in the PFC of BTBR mice, indicating processes may evade miRNA control. In contrast, other miRNAs were predicted to have extensive relationships with DEGs suggesting their role as potential hub coordinators of gene expression. Profiling findings were confirmed via qRT-PCR for representative protein-coding transcripts and miRNAs. Our study underscores the complex interplay between gene expression and miRNA regulation within immune and inflammatory pathways in the BTBR model, offering insights into the neurodevelopmental mechanisms of ASD. These results support the value of the BTBR mouse model and identify strategies that could adjust molecular pathways for therapeutic applications in ASD research.

Keywords: Autism spectrum disorder; Inflammatory pathways; MRNA; MiRNA; Prefrontal cortex.

Fig. 1

Hierarchical clustering representation and overrepresented pathway categories in the PFC of BTBR mice. a Heatmap showing hierarchical clustering of 1063 differentially expressed genes in the prefrontal cortex between BTBR and B6 control mice. A color-coded scale was used to show gene expression differences in logarithmic fold change units between the groups (blue represents the lowest expression; yellow represents the highest expression)(b-d) Overrepresented pathway categories for differentially expressed genes in the BTBR PFC: all DEGs (b), up-regulated (Up) DEGs (c), and down-regulated (Dw) DEGs (d). DEGs were analysed for enrichment in pathway categories using DAVID, with a p-value < 0.05. For each term, the number of genes is indicated by the length of horizontal bars (gene counts).

Fig. 2

Mouse phenotype categories and enrichment analysis on BTBR PFC DEGs. (a) PFC BTBR DEGs were analyzed for enrichment in mouse phenotypes using Enrichr with a p-value cutoff of 0.05. The top five significant phenotype categories found in the BTBR DEGs are arranged from the most to the least significant. The number of genes in the category is indicated by the length of the horizontal bars (gene counts). (b) Up- and downregulated DEGs and DEmiRNAs were subjected to enrichment analysis for cell-type specific markers, neurodevelopmental and neurodegenerative disease terms, and transcriptomic datasets derived from patients or mouse models of neurodevelopmental disorders. Enrichment significance is represented by color, with white for p > 0.05 and gray for p < 0.05

Fig. 3

Heat map of DEmiRNAs and KEGG pathway enrichment analysis of predicted miRNA targets. (a) Heat map of the 48 differentially expressed miRNAs, with unsupervised hierarchical clustering by miRNAs, in the prefrontal cortex between BTBR and B6 mice. The dendrogram was constructed using the complete linkage method with the Euclidean distance measure. The analysed samples are shown in columns, and the DEmiRNAs are presented in rows. Yellow represents increased expression and blue represents reduced expression. (b) KEGG pathway analysis of the predicted target genes for up- and down-regulated DEmiRNAs in the PFC between the BTBR and B6 control mice. DEmiRNA target genes were analysed for enrichment in KEGG pathways using DAVID with a p-value cutoff of 0.05. The x-axis shows the counts of DEmiRNA target genes enriched in KEGG pathways, and the y-axis shows the KEGG pathways. Yellow bars represent the target genes of upregulated miRNAs, while blue those of down-regulated miRNAs. KEGG pathways are ranked from top to bottom based on the difference between down- and up-regulated miRNA gene targets in the ratio of gene counts to the number of genes in the pathway (Population Hits)

Fig. 4

Balloon plot representing the most significant results of g:Profiler analysis onDEmiRNAs. The x-axis distinguishes between down- and upregulated miRNAs, while the y-axis lists significant biological terms. The size of the balloons is proportional to the number of miRNAs involved in each biological pathway, and the color gradient and intensity reflect the significance of the enrichment, represented by the negative logarithm of the corrected p-value

Fig. 5

DAVID pathway analysis for upregulated DEGs and target genes of downregulated DEmiRNAs. Only pathways with a significant p-value (< 0.05) that were shared between the two groups were considered. For each pathway, the number of genes is indicated by the length of horizontal bars (gene counts). Yellow bars represent upregulated DEGs, while blue bars indicate target genes of downregulated DEmiRNAs

Fig. 6

IPA functional analysis for (a) downregulated DEGs and target genes of upregulated DEmiRNAs and (b) upregulated DEGs and target genes of downregulated DEmiRNAs. Only pathways with a significant adjusted p-value (< 0.05) that were shared between the two groups were considered. Yellow balloons indicate upregulation, while blue balloons indicate downregulation. For each function, reported on the x-axis, the balloon size is proportional to the negative base 10 logarithm of the adjusted p-value (-log10(P))

Fig. 7

g:Profiler functional analysis for (a) downregulated DEGs and target genes of upregulated DEmiRNAs and (b) upregulated DEGs and target genes of downregulated DEmiRNAs. Only pathways shared between the two groups and with a significant adjusted p-value (< 0.05) were considered. For each biological term, reported on the y-axis, the size of the balloons is proportional to the number of genes involved in the biological pathway, while the color gradient and intensity reflect the significance of the enrichment, represented by the negative logarithm of the corrected p-value

Fig. 8

DEmiRNA-DEG relation analysis. Pie charts illustrate the percentage and size of unique relationship retrieved with IPA between different combinations of up- and downregulated DEGs and DEmiRNAs. (a) The left chart shows results using the shortest path between nodes. (b) The right chart includes the shortest path plus one additional intervening node. The tables below the pie charts summarize the size of the relationships and the chi-square significance values

Fig. 9

(a) qRT-PCR validation of DEGs. qRT-PCR results for all the evaluated genes were consistentwith microarray results. Values are expressed as comparative quantitation ratios (gene/L41), normalized to the expression levels of controls (mean ± S.E.M. of three replicates from pools of six animals per genotype; p < 0.05, Student’s t-test, BTBR vs. B6). (b) qRT-PCR validation of DEmiRNAs. qRT-PCR results for all the evaluated DEmiRNAs were consistent with open array results. Values are expressed as comparative quantitation ratios (miRNA/RNU19), normalized to the expression levels of controls (mean ± s.e.m. of three replicates from pools of six animals per genotype; p < 0.05, Student’s t-test, BTBR vs. B6).