Transcriptomic and epigenomic profiling reveals altered responses to diesel emissions in Alzheimer's disease both in vitro and in population-based data

Abstract

Introduction: Studies have correlated living close to major roads with Alzheimer's disease (AD) risk. However, the mechanisms responsible for this link remain unclear.

Methods: We exposed olfactory mucosa (OM) cells of healthy individuals and AD patients to diesel emissions (DE). Cytotoxicity of exposure was assessed, mRNA, miRNA expression, and DNA methylation analyses were performed. The discovered altered pathways were validated using data from the human population-based Rotterdam Study.

Results: DE exposure resulted in an almost four-fold higher response in AD OM cells, indicating increased susceptibility to DE effects. Methylation analysis detected different DNA methylation patterns, revealing new exposure targets. Findings were validated by analyzing data from the Rotterdam Study cohort and demonstrated a key role of nuclear factor erythroid 2-related factor 2 signaling in responses to air pollutants.

Discussion: This study identifies air pollution exposure biomarkers and pinpoints key pathways activated by exposure. The data suggest that AD individuals may face heightened risks due to impaired cellular defenses.

Highlights: Healthy and AD olfactory cells respond differently to DE exposure. AD cells are highly susceptible to DE exposure. The NRF2 oxidative stress response is highly activated upon air pollution exposure. DE-exposed AD cells activate the unfolded protein response pathway. Key findings are also confirmed in a population-based study.

Keywords: Alzheimer's disease (AD); air pollution; air–liquid interface (ALI); heat shock protein (HSP); next‐generation sequencing (NGS); nuclear factor erythroid 2–related factor 2 (NRF2); traffic emissions; traffic‐related air pollution (TRAP) olfactory mucosa (OM).

FIGURE 1

Transcriptomic changes in control and AD OM cells caused by DE exposure. Common and top deregulated genes. (A) List of common significant DEGs in both control and AD cells after DE exposure (p‐adjusted < 0.05) sorted by decreasing logFC. Venn diagram showing number of DEGs in AD and control cells after exposure. (B) Heatmap showing expression value for most deregulated genes (highest absolute logFC value) after DE exposure (p‐adjusted < 0.05). AD, Alzheimer's disease; DE, diesel emissions; DEG, differentially expressed gene.

FIGURE 2

Induction of oxidative stress response in control and AD OM cells after DE exposure. Volcano plot highlighting the most altered oxidative stress genes after DE exposure in control (A) and AD (B) OM cells. Here, we labeled on the plot genes that were previously reported in a Gene Ontology biological process related to oxidative stress. The y‐axis of the volcano graph is ‐log10 (p value), the x‐axis is the logFC value; red: upregulated genes, blue: downregulated genes. (C) Heatmap of top‐upregulated oxidative stress‐related genes induced by DE exposure in AD cells (left) and control cells (right). The asterisk indicates the genes significant in the category. On top is indicated the number of significant genes that cause an enrichment in oxidative stress. AD, Alzheimer's disease; DE, diesel emissions; OM, olfactory mucosa.

FIGURE 3

Differentially expressed miRNA–mRNA relationships in (A) control and (B) AD OM cells after DE exposure. Specifically, the anticorrelated relationships between miRNA and genes of some relevant pathways are depicted. Pathways investigated relate to oxidative stress response (GO:0006979 and GO:0034599) and response to unfolded protein or protein folding in the ER (GO:006986 and GO:0034975). The color scale corresponds to the logFC expression of miRNA and mRNA, the triangle‐shaped nodes represent miRNA, while circles are mRNAs. Colored borders of nodes indicate the target genes involved in the respective pathways. AD, Alzheimer's disease; DE, diesel emissions; ER, endoplasmic reticulum; OM, olfactory mucosa.

FIGURE 4

(A) Top canonical pathways activated by DE exposure in OM cells provided by IPA software, sorted by z‐score value. (B) Top significant transcription factors, sorted by activation value (z‐score). (C) Heatmap showing NRF2‐mediated oxidative stress response genes affected by DE exposure in control and AD OM cells. The asterisk indicates the genes significant in the category and that were used in the enrichment. On top is indicated the number of significant genes that cause an enrichment in oxidative stress. AD, Alzheimer's disease; DE, diesel emissions; IPA, Ingenuity Pathway Analysis; NRF2, nuclear factor erythroid 2–related factor 2; OM, olfactory mucosa.

FIGURE 5

Activation of UPR pathway in AD OM cells after DE exposure calculated with IPA software. AD, Alzheimer's disease; DE, diesel emissions; IPA, Ingenuity Pathway Analysis; OM, olfactory mucosa; UPR, unfolded protein response.

FIGURE 6

(A) HSP70 level is increased in AD OM cell media after DE exposure. Quantification of HSP70 was performed with ELISA and t‐test (unpaired, two‐tailed, two‐way ANOVA with Sidak multiple test). Data are presented as mean ± SD, **p < .01. (B) Graphical representation of number of DMRs and relative methylation change (hyper vs hypo) after DE exposure in control and AD OM cells. Figures represent correlation of DMRs with mRNA expressions altered in control OM cells (C) and AD OM cells (D) after DE exposure. AD, Alzheimer's disease; DE, diesel emissions; DMR, differentially methylated region; HSP70, heat shock protein 70; IPA, Ingenuity Pathway Analysis; OM, olfactory mucosa.

FIGURE 7

Number of DEGs in DE‐exposed AD OM cells (A) and DE‐exposed control OM cells (B) found in representative pathways of highly polluted areas whose residents participated in Rotterdam Study. Venn diagram represents overlap of the two pathway lists. AD, Alzheimer's disease; DE, diesel emissions; DEG, differentially expressed gene; OM, olfactory mucosa.