Microbiome drives age-dependent shifts in brain transcriptomic programs at the single-cell level in Drosophila

Abstract

The gut microbiome plays a critical role in brain function and the brain-gut axis, yet its cellular and molecular mechanisms remain unclear. Here, we present the first comprehensive single-cell transcriptomic atlas of brain cells from adult Drosophila melanogaster raised under axenic and microbiome-associated conditions, spanning young and old ages. Profiling 34,427 cells across 101 clusters, we annotated 56 cell types and identified cell type-specific gene signatures influenced by the microbiome. Transcriptional shifts were most pronounced in old flies, with glial cells and dopaminergic neurons among the most microbiome-responsive cell types. Differentially expressed genes (DEGs) were enriched in pathways related to mitochondrial activity, energy metabolism, and Notch signaling. We also quantified age-associated changes in the gut microbiome, observing reduced Acetobacter dominance and increased microbial diversity that corresponded with heightened brain transcriptional responses. These findings illuminate the cell type-specific impacts of the microbiome on brain gene expression and lay the groundwork for understanding the molecular underpinnings of the microbiome-gut-brain axis.

Fig. 1. Single-cell transcriptomic profiling of the Drosophila brain across different bacterial conditions and ages.

a Sampling of Drosophila brains from adult microbiome-associated and axenic flies (female) at two distinct ages. b t-distributed stochastic neighbor embedding (t-SNE) visualization of cells from all treatment groups. c Identified cell types from single-cell RNA-seq clustering analysis: (Top) dendrogram illustrating the hierarchical relationships among identified cell types; (Bottom) expression levels and proportions of selected marker genes for identified cell types.

Fig. 2. Microbiome-responsive DEGs across brain cell types between young and old flies.

a The Π_g index, calculated for each DEG (microbiome-associated group vs. axenic group) with FDR < 0.05, quantifies the weighted impact of differential expression. Each dot represents a DEG. The color gradient indicates the detection rate, reflecting the proportion of cell types in which a DEG was significantly detected. A linear regression line (solid red) indicates the relationship between Πg in the two age groups. b Heatmap of 20 DEGs with the highest Π_g values in each age group. Only annotated cell types and protein-coding genes are shown.

Fig. 3. Gene set enrichment analysis of Π_g.

Gene set enrichment analysis (GSEA) was performed using ranked lists of Π_g separately for each age group. The top 20 enriched gene sets with the highest normalized enrichment scores (NES) are displayed separately in each domain, along with their corresponding adjusted p-values.

Fig. 4. Global and DEG-based transcriptional responses to the gut microbiome across brain cell types.

The impact of the gut microbiome on each brain cell type was assessed using two metrics: a global transcriptome shift and b Πc of all differentially expressed genes (DEGs) with FDR < 0.05. Log-transformed Manhattan distances of gene expression fold changes, and Πc values were calculated separately for microbiome-associated and axenic flies in young and old groups. Each dot represents a cell type. The red regression line indicates the linear relationship between young and old flies, with corresponding statistics shown at the bottom. c Rankings of brain cell types in old flies based on global transcriptome shifts and Πc values. The top 20 cell types with the highest values in each metric are circled. Three cell types—T1 neurons, dopaminergic neurons, and subperineurial glia—appear in the top 20 for both metrics and are highlighted in red.

Fig. 5. Gut Bacterial community structures in young and old flies.

a Relative abundance of bacterial species in young and old flies quantified by 16S rRNA gene amplicon sequencing. b Comparison of alpha diversity metrics (Observed number of genera and Shannon Index) between the age groups. c Quantification of culturable bacteria from the gut on LB and MRS agar plates for young and old flies.