Single Nuclei Transcriptomics Reveals Obesity-Induced Endothelial and Neurovascular Dysfunction: Implications for Cognitive Decline

Abstract

Obesity confers risk for cardiovascular disease and vascular dementia. However, genomic alterations modulated by obesity in endothelial cells in the brain and their relationship to other neurovascular unit (NVU) cells are unknown. We performed single nuclei RNA sequencing (snRNAseq) of the NVU (endothelial cells, astrocytes, microglia, and neurons) from the hippocampus of obese (ob/ob) and wild-type (WT) male mice to characterize obesity-induced transcriptomic changes in a key brain memory center and assessed blood-brain barrier permeability (BBB) by gadolinium-enhanced magnetic resonance imaging (MRI). Ob/ob mice displayed obesity, hyperinsulinemia, and impaired glucose tolerance. snRNAseq profiled 14 distinct cell types and 32 clusters within the hippocampus of ob/ob and WT mice and uncovered differentially expressed genes (DEGs) in all NVU cell types, namely, 4462 in neurons, 1386 in astrocytes, 125 in endothelial cells, and 154 in microglia. Gene ontology analysis identified important biological processes such as angiogenesis in endothelial cells and synaptic trafficking in neurons. Cellular pathway analysis included focal adhesion and insulin signaling, which were common to all NVU cell types. Correlation analysis revealed significant positive correlations between endothelial cells and other NVU cell types. Differentially expressed long non-coding RNAs (lncRNAs) were observed in cells of the NVU-affecting pathways such as TNF and mTOR. BBB permeability showed a trend toward increased signal intensity in ob/ob mice. Taken together, our study provides in-depth insight into the molecular mechanisms underlying cognitive dysfunction in obesity and may have implications for therapeutic gene targeting.

Keywords: hippocampus; neurovascular unit; obesity; single nuclei transcriptomics.

Figure 1

Single nuclei RNA-sequencing identifies global genomic cell-specific changes in the hippocampus of obese mice. (A) Overview of this study. Hippocampi were isolated from normal weight (wild type) and from obese (ob/ob) male mice (n = 4/genotype) at 18 weeks of age. Single nucleotide RNA sequencing was performed on isolated hippocampi, followed by in-depth bioinformatics analyses. (B) Uniform manifold approximation and projection (UMAP) showing the cell clusters in hippocampal cells in obese and normal-weight mice. (C) Schematic presentation of the major cells of the neurovascular unit (NVU) created in biorender.com (accessed on 27 February 2024). (D) Sparse Partial Least Squares Discriminant Analysis (sPLS-DA) performed using normalized global gene expression datasets from NVU cell types. (E) Heatmap of normalized gene expression, where the rows are expressed genes and the columns are the individual samples, grouped by cell types of NVU. Red denotes higher levels of gene expression, and blue denotes lower levels of gene expression, as indicated in the color bar.

Figure 2

Obesity significantly changes gene expression in endothelial, microglial, astrocytes, and neuronal cells of the hippocampus. (A) Volcano plots show differential gene expression for ob/ob mice compared to control WT mice for the four cell types of NVU. Only significantly changed gene expressions (p < 0.06) are colored according to the direction of change, that is, blue for downregulation and red for upregulation of gene expression. (B) Bar plot showing the number of differentially expressed genes (DEGs) both downregulated (green) and upregulated (red) across the four cell types of the NVU. (C) Distribution of fold changes for significant DEGs between obese and normal-weight mice across NVU cell populations. (D) Venn diagram of DEGs in endothelial cells (EC), microglial cells (MG), astrocytes (AS), and neurons (NEU) showing overlaps between sets of identified genes. (E) Violin plots of the expression levels presented as the normalized counts of common DEGs across cell types of the NVU (EC, MG, AS, and NEU).

Figure 3

Functional classification of DEGs identifies cell-specific and common major cellular pathways regulating cell–cell interactions, metabolism, immune system, cell signaling, and neurofunction across cells of the NVU. (A) Venn diagram comparing identified significantly enriched pathways for the four cell types of the NVU with DEGs altered by obesity compared to normal-weight mice. (B) Dot plot of cell–cell interaction pathways regulated by DEGs in the NVU cell types. Significant pathways are presented with large blue circles. A heatmap representing expression levels of genes involved in focal adhesion across the NVU cells is presented on the right. (C) Dot plot of immune system pathways modulated by DEGs in the NVU cell types. (D) Dot plot of pathways of DEGs involved in the regulation of metabolism in the NVU cells. A heatmap representing expression levels of genes involved in the insulin signaling pathway across the NVU cell types is presented on the right. (E) Dot plot of pathways of DEGs involved in cell signaling in the NVU cells. (F) Dot plot of pathways of DEGs involved in the neurofunction regulation in the NVU cell types.

Figure 4

Positive correlation of global gene expression changes by obesity in endothelial cells with other cell types of the hippocampal NVU. (A) Scatter plots of genes showing significant (p < 0.05) positive correlation between endothelial cells and microglia cells (r = 0.15), endothelial cells and astrocytes (r = 0.19), and endothelial cells and neuronal cells (r = 0.16). (B) Gene–gene correlation matrices of genes identified as differentially expressed in endothelial cells and microglia cells, endothelial cells and astrocytes, and endothelial cells and neuronal cells. For each gene–gene correlation, positive correlation is presented in blue and negative correlation in red; significant (p < 0.05) correlations are presented with “*”.

Figure 5

Obesity induces cell-specific changes in the expression of long non-coding RNAs. (A) Table with identified differentially expressed (DE) long non-coding RNAs (lncRNAs) specific to each cell type and in common among the cells of NVU. (B) Pathway enrichment analysis of target genes of the 20 most significant DE lncRNAs in the NVU cell types. The colored Venn diagram shows pathways specific to endothelial cells (EC) and in common to other NVU cell types (NEU: neuronal cells; AS: astrocytes; MG: microglia).

Figure 6

Obesity modulates the expression of genes in NVU cell types associated with neurodegenerative diseases. (A) Venn diagram showing the number of significant associations between DEGs of NVU cell types and neurodegenerative diseases. (B) Network representing interactions between genes differentially expressed by obesity in the NVU cell types with Alzheimer’s disease, cerebrovascular diseases, and dementia. DEGs in endothelial cells (e, green circles), astrocytes (a, blue circles), neuronal cells (n, yellow circles), and microglial cells (m, pink circles).

Figure 7

Obesity predisposes to increased blood–brain barrier permeability. (A) Representative magnetic resonance imaging (MRI) images of the brain pre- and post-gadolinium (Gd) infusion for ob/ob and WT mice and percent difference in intensity. (B) Dynamic contrast enhanced (DCE) plots showing relative signal intensity (y-axis) over time (x-axis). (C) Area under the cure (AUC) of the DCE plot for ob/ob and WT mice (n = 8–9/genotype).

Figure 8

Summary schematic of the effect of obesity on the cellular functions of the hippocampal neurovascular unit and implications for cognitive decline.