Disease-associated astrocytes and microglia markers are upregulated in mice fed high fat diet

Abstract

High-fat diet (HFD) is associated with Alzheimer's disease (AD) and type 2 diabetes risk, which share features such as insulin resistance and amylin deposition. We examined gene expression associated with astrocytes and microglia since dysfunction of these cell types is implicated in AD pathogenesis. We hypothesize gene expression changes in disease-associated astrocytes (DAA), disease-associated microglia and human Alzheimer's microglia exist in diabetic and obese individuals before AD development. By analyzing bulk RNA-sequencing (RNA-seq) data generated from brains of mice fed HFD and humans with AD, 11 overlapping AD-associated differentially expressed genes were identified, including Kcnj2, C4b and Ddr1, which are upregulated in response to both HFD and AD. Analysis of single cell RNA-seq (scRNA-seq) data indicated C4b is astrocyte specific. Spatial transcriptomics (ST) revealed C4b colocalizes with Gfad, a known astrocyte marker, and the colocalization of C4b expressing cells with Gad2 expressing cells, i.e., GABAergic neurons, in mouse brain. There also exists a positive correlation between C4b and Gad2 expression in ST indicating a potential interaction between DAA and GABAergic neurons. These findings provide novel links between the pathogenesis of obesity, diabetes and AD and identify C4b as a potential early marker for AD in obese or diabetic individuals.

Figure 1

Experimental workflow. (A) The workflow of RNA-seq analysis. (B) The procedures of RNA-seq data generation from the mouse model of HFD-induced obesity and diabetes.

Figure 2

Heatmaps and volcano plots of up- and down- regulated genes in brains from mice fed HFD versus a regular diet. (A) DEGs identified in dorsal preoptic brain region are displayed in a heatmap (FDR-adjusted p-value < 0.05 and |log2 FC|> 0.63). (B) DEGs identified in dorsal preoptic brain region are displayed in a volcano plot (FDR-adjusted p-value < 0.05 and |log2 FC|> 0.3). A heatmap (C) and a volcano plot (D) show DEGs identified in the ventral preoptic region brain region (FDR adjusted p-value < 0.05 and |log2 FC|> 0.3). Note that in the heatmaps the control samples are in the left three columns, while the right five columns represent samples from mice on HFD. The color scales in heatmps (A) (C) represent normalized expression levels with performing pheatmap. In the heatmaps and volcano plots, up-regulated genes are shown in red, while down-regulated genes are shown in blue.

Figure 3

Hierarchical clustering heatmaps, correlation heatmaps and theoretical regulation of gene expression in the preoptic region. (A, B) Heatmaps showing the expression profile of 15 DEGs identified from HFD cohort in dorsal preoptic region (A) and ventral preoptic region (B) overlapping with DAA, DAM and HAM markers. The left 3 columns represent 3 control samples and the rest columns on the right are samples from HFD. (C) A heatmap for 11 common DEGs found among DAA, DAM and HAM markers, HFD and AD brain samples. The red and the blue color represent up-regulated or down-regulated genes respectively. The color scales in heatmaps (A) (B) (C) represent normalized expression levels with performing pheatmap. (D) Correlation heatmap using Pearson method between DEGs and the clinical phenotypes with Pearson’s correlation coefficient in each cell between DEGs and AD clinical phenotypes. The color scale displays Pearson correlation. Red color indicates positive correlation while blue color shows negative correlation. The darker the color means the stronger correlation. (E) The plots illustrate the theoretical changes in genes from consumption of high-fat diet to the development of AD.