Role of caveolin-1 in metabolic programming of fetal brain

Abstract

Mice lacking caveolin-1 (Cav1), a key protein of plasma membrane, exhibit brain aging at an early adult stage. Here, integrative analyses of metabolomics, transcriptomics, epigenetics, and single-cell data were performed to test the hypothesis that metabolic deregulation of fetal brain due to the ablation of Cav1 is linked to brain aging in these mice. The results of this study show that lack of Cav1 caused deregulation in the lipid and amino acid metabolism in the fetal brain, and genes associated with these deregulated metabolites were significantly altered in the brain upon aging. Moreover, ablation of Cav1 deregulated several metabolic genes in specific cell types of the fetal brain and impacted DNA methylation of those genes in coordination with mouse epigenetic clock. The findings of this study suggest that the aging program of brain is confounded by metabolic abnormalities in the fetal stage due to the absence of Cav1.

Keywords: Bioinformatics; Metabolomics; Neuroscience; Omics; Transcriptomics.

Graphical abstract

Figure 1

Experimental design and metabolomics analysis (A) Timed pregnancy was performed separately with WT and Cav1-null mice to collect GD15 fetal brain. (B) Pairwise cluster analysis of variation of metabolite levels show that specific lipids, including cholesterol, were suppressed whereas several amino acids were activated in the fetal brain due to the absence of Cav1.

Figure 2

Integrative analysis of metabolomics and RNA-seq data (A) Metabolites (Ms) were mapped to KEGG compounds database, and genes (Gs) were mapped to KEGG genes database to identify metabolite-gene pairs that mapped to the same pathways (Ps). (B) The direction of change of metabolite and gene was then evaluated. Pairs in which both the gene and metabolite showed similar change (U: upregulation, D: downregulation) in the brain of Cav1-null compared to WT mice were extracted. They were subjected to canonical correlation analysis. (C) Canonical changes of metabolites (U: upregulated, D: downregulated) clustered into three distinct clusters (CL1, CL2, and CL3). Each dot here represents a metabolite. The patterns of variation are shown in boxplots with different colors. (D) Canonical changes of genes (U: upregulated, D: downregulated) in the same three clusters (CL1, CL2, and CL3). Each dot represents a gene. The patterns of variation are shown in boxplots with different colors.

Figure 3

Epigenetic changes of fetal brain and influence on gene expression of aging brain (A) Higher level of methylation (beta values, shown in y axis) in Cav1-null (KO) brain compared to WT brain (female:1, male:2). (B) Lower level of methylation in Cav1-null brain compared to WT brain (female:1, male:2). (C) In the middle panel, Venn diagram shows the number of methylations (shown as “m”) identified in clock genes (shown as “g”). The number of methylations that increased in the fetal brain of Cav1-null compared to WT mice is shown in red. The genes are shown as gray bar with red stars (methylations). An opposite pattern was also observed where CpG sites were hypo-methylated in the fetal brain of Cav1-null compared to WT mice. The genes are shown as gray bar with blue stars (methylations). In a third group of genes (n = 60), both the types of methylations were observed. They are shown as gray bar (gene) with both red and blue stars (methylations). Specific metabolic genes were identified within these three groups of differentially methylated genes. The gene name, number of methylations (within parenthesis) in the gene, and association with deregulated metabolites are shown.

Figure 4

Dimensional plot of single-cell gene expression of Cav1-null and WT fetal brain PC1 and PC2 represent the principal component axes. Each dot represents a cell. Cell types are color coded as shown in the legends.

Figure 5

Cell types and metabolic genes as cell specific markers In the upper panel, the histograms show the differential abundance of glial and neuronal cells in the fetal brain of WT (A) and Cav1-null mice (B). In the lower panel, the heatmaps show association of identified marker genes with lactic acid (LA), L-tyrosine (TY), myo-inositol (MI), glycerophosphate ester (GP), L-glutamic acid (GA), and cholesterol (CH) in different cell types of WT (C) and Cav1-null fetal brain (D). The scales right to the heatmaps show the color codes for the number of marker genes.

Figure 6

Violin plots showing expression of Runx1t1 and Cntnap2 in brain cells of WT (A) and Cav1-null mice (B) X axis shows the cell type and Y axis shows the relative expression values of the integrated data. The cell types in the X axis are abbreviated as R (radial glia), E (ependymal), M (microglia), N (neuron) for both the wild-type (WT) and knockout (KO) samples. The kernel density, wherever applicable, is shown with color.