Longitudinal APOE4- and amyloid-dependent changes in the blood transcriptome in cognitively intact older adults

Abstract

Background: Gene expression is dysregulated in Alzheimer's disease (AD) patients, both in peripheral blood and post mortem brain. We investigated peripheral whole-blood gene (co)expression to determine molecular changes prior to symptom onset.

Methods: RNA was extracted and sequenced for 65 cognitively healthy F-PACK participants (65 (56-80) years, 34 APOE4 non-carriers, 31 APOE4 carriers), at baseline and follow-up (interval: 5.0 (3.4-8.6) years). Participants received amyloid PET at both time points and amyloid rate of change derived. Accumulators were defined with rate of change ≥ 2.19 Centiloids. We performed differential gene expression and weighted gene co-expression network analysis to identify differentially expressed genes and networks of co-expressed genes, respectively, with respect to traits of interest (APOE4 status, amyloid accumulation (binary/continuous)), and amyloid positivity status, followed by Gene Ontology annotation.

Results: There were 166 significant differentially expressed genes at follow-up compared to baseline in APOE4 carriers only, whereas 12 significant differentially expressed genes were found only in APOE4 non-carriers, over time. Among the significant genes in APOE4 carriers, several had strong evidence for a pathogenic role in AD based on direct association scores generated from the DISQOVER platform: NGRN, IGF2, GMPR, CLDN5, SMIM24. Top enrichment terms showed upregulated mitochondrial and metabolic pathways, and an exacerbated upregulation of ribosomal pathways in APOE4 carriers compared to non-carriers. Similarly, there were 33 unique significant differentially expressed genes at follow-up compared to baseline in individuals classified as amyloid negative at baseline and positive at follow-up or amyloid positive at both time points and 32 unique significant differentially expressed genes over time in individuals amyloid negative at both time points. Among the significant genes in the first group, the top five with the highest direct association scores were as follows: RPL17-C18orf32, HSP90AA1, MBP, SIRPB1, and GRINA. Top enrichment terms included upregulated metabolism and focal adhesion pathways. Baseline and follow-up gene co-expression networks were separately built. Seventeen baseline co-expression modules were derived, with one significantly negatively associated with amyloid accumulator status (r² = - 0.25, p = 0.046). This was enriched for proteasomal protein catabolic process and myeloid cell development. Thirty-two follow-up modules were derived, with two significantly associated with APOE4 status: one downregulated (r² = - 0.27, p = 0.035) and one upregulated (r² = 0.26, p = 0.039) module. Top enrichment processes for the downregulated module included proteasomal protein catabolic process and myeloid cell homeostasis. Top enrichment processes for the upregulated module included cytoplasmic translation and rRNA processing.

Conclusions: We show that there are longitudinal gene expression changes that implicate a disrupted immune system, protein removal, and metabolism in cognitively intact individuals who carry APOE4 or who accumulate in cortical amyloid. This provides insight into the pathophysiology of AD, whilst providing novel targets for drug and therapeutic development.

Keywords: APOE4; Alzheimer’s disease; Amyloid accumulation; Blood; Longitudinal study; RNA sequencing; Transcriptome.

Fig. 1

Amyloid change in F-PACK participants, stratified for APOE4 status. The dotted line represents the threshold for amyloid positivity = 23.5. Red = APOE4 carrier (N = 31), blue = APOE4 non-carrier (N = 34)

Fig. 2

Differentially expressed genes at follow-up compared to baseline stratified for APOE4 or amyloid positivity status. A Volcano plot showing the differentially expressed genes at follow-up compared to baseline in APOE4 carriers. B Volcano plot showing the differentially expressed genes at follow-up compared to baseline in APOE4 non-carriers. C Volcano plot showing the differentially expressed genes at follow-up compared to baseline in individuals amyloid positive at both time points or amyloid negative at baseline, positive at follow-up. D Volcano plot showing the differentially expressed genes at follow-up compared to baseline in individuals amyloid negative at both time points. Data points are coloured based on significance: grey = non-significant, blue = non-significant but with p_FDR-value < 0.05, red = significant with p_FDR-value < 0.05 and log2FoldChange ± 1. APOE4 carriers N = 31, APOE4 non-carriers N = 34. Amyloid positive-positive or amyloid negative–positive N = 9, amyloid negative-negative N = 56

Fig. 3

WGCNA of baseline expression data. A Correlation heatmap of traits of interest with WGCNA modules depicting the Pearson’s correlation coefficient with the p-value in brackets. B Gene ontology enrichment terms derived from over-representation analysis. C Protein–protein interaction network of highly interconnected genes with hub genes shaped as hexagonal

Fig. 4

WGCNA of follow-up expression data. A Correlation heatmap of traits of interest with WGCNA modules depicting the Pearson’s correlation coefficient and the p-value in brackets. B Gene ontology enrichment terms derived from over-representation analysis for the blue module. C Protein–protein interaction network of highly interconnected genes with hub genes shaped as hexagonal for the blue module. D Gene ontology enrichment terms derived from over-representation analysis for the turquoise module. E Protein–protein interaction network of highly interconnected genes with hub genes shaped as hexagonal for the turquoise module. Note the differing sliding colour scale for the enrichment plots

Fig. 5

Module preservation and consensus of baseline or follow-up co-expression modules. Zsummary scores of A baseline WGCNA modules in follow-up expression data and B follow-up co-expression modules in baseline expression data. A Zsummary < 2 suggests low preservation (below blue dotted line); > 2 Zsummary < 10 suggests moderate preservation; Zsummary > 10 suggests high preservation (above green dotted line). Correspondence of C baseline set-specific modules or D follow-up set-specific modules with consensus modules. Each row is the set-specific module and each column is a consensus module. The more red the cell, the more significant the Fisher’s exact p-value (encoded by -log(p) signifying the gene overlap. For module labels in plots A and B for the preservation analyses please see y-axes module