Proteomics analysis of plasma from middle-aged adults identifies protein markers of dementia risk in later life

Abstract

A diverse set of biological processes have been implicated in the pathophysiology of Alzheimer's disease (AD) and related dementias. However, there is limited understanding of the peripheral biological mechanisms relevant in the earliest phases of the disease. Here, we used a large-scale proteomics platform to examine the association of 4877 plasma proteins with 25-year dementia risk in 10,981 middle-aged adults. We found 32 dementia-associated plasma proteins that were involved in proteostasis, immunity, synaptic function, and extracellular matrix organization. We then replicated the association between 15 of these proteins and clinically relevant neurocognitive outcomes in two independent cohorts. We demonstrated that 12 of these 32 dementia-associated proteins were associated with cerebrospinal fluid (CSF) biomarkers of AD, neurodegeneration, or neuroinflammation. We found that eight of these candidate protein markers were abnormally expressed in human postmortem brain tissue from patients with AD, although some of the proteins that were most strongly associated with dementia risk, such as GDF15, were not detected in these brain tissue samples. Using network analyses, we found a protein signature for dementia risk that was characterized by dysregulation of specific immune and proteostasis/autophagy pathways in adults in midlife ~20 years before dementia onset, as well as abnormal coagulation and complement signaling ~10 years before dementia onset. Bidirectional two-sample Mendelian randomization genetically validated nine of our candidate proteins as markers of AD in midlife and inferred causality of SERPINA3 in AD pathogenesis. Last, we prioritized a set of candidate markers for AD and dementia risk prediction in midlife.

Fig. 1.. Proteome-wide association study for 25-year dementia risk.

Hazard ratios (HRs) for all analyses were derived from Cox proportional hazards regression models adjusted for age, sex, race-study center, education, APOEϵ4 status, and estimated glomerular filtration rate (eGFR) creatinine, body mass index, diabetes, hypertension, and smoking status at the time of protein assessment. (A) Volcano plot displays the HRs (x axis) and two-sided P values (y axis) for the association of log₂ protein abundance with incident dementia. Proteins above the horizontal red line maintained a significant association after Bonferroni correction. (B) The majority of dementia-associated proteins were implicated in one of six biological pathways based on associated Gene Ontology terms. (C) Venn diagram shows candidate dementia-associated proteins from analysis of full-term, near-term, and long-term dementia risk. (D) Volcano plot displays the association of log₂ protein abundance with incident dementia occurring within 15 years of follow-up (near-term dementia). (E) Volcano plot displays the association of log₂ protein abundance with incident dementia occurring beyond 15 years of follow-up (long-term dementia). (F) HRs for all 32 dementia-associated proteins in an analysis of near-term dementia risk (x axis) plotted against HRs from an analysis of long-term dementia risk (y axis). Color indicates in which analyses proteins were found to be statistically significant at a proteome-wide significance threshold. (G) This figure compares HRs from the primary analyses with HRs derived from participants below age 60 at the time blood was drawn for protein measurement. To make HRs directly comparable to HRs derived from the primary analysis, the six proteins associated with near-term dementia risk were examined in relation to dementia occurring within 15 years (n = 5285; 66 dementia cases). All other proteins were examined using the full follow-up time (n = 5285; 525 dementia cases).

Fig. 2.. Protein-neurologic disease/trait gene overlap and enriched biological pathways for dementia-associated proteins.

(A) Proteins significantly associated with dementia risk (FDR-corrected P < 0.05) and coded for by genes linked to GWAS risk variants for neurodegenerative and psychiatric disease, intelligence, and cognition. Gene lists were based on GWAS catalog summary statistics [by Yang and colleagues (49)] and recent AD GWAS (14). Bolded proteins were associated with dementia risk at a Bonferroni-corrected threshold. (B) Canonical (biological) pathways were identified using Ingenuity Pathway Analysis (IPA). The top 25 pathways for each analysis are displayed. Statistical significance was defined as an FDR-corrected P < 0.05 (one-sided) using right-tailed Fisher’s exact test. The threshold for statistical significance is represented by the vertical dotted line. Number in each bar is a Z-score which indicates the predicted degree of pathway activation or inhibition. The direction of activation could not be predicted for gray bars. The extent of activation could not be predicted for bars with no Z-scores. Results presented in the top row are derived using the full set of SomaScan proteins included in the study as a reference gene set. PDGF, platelet-derived growth factor; HIF-1α, hypoxia-inducible factor–1α. (C) Results presented in the second row are derived using the full gene list in the IPA database as the reference gene set. AKT, protein kinase B; ALS, amyotrophic lateral sclerosis; ARE, AU-rich element; BAG2, bcl2-associated athanogene 2; CAR, chimeric antigen receptor; GP6, glycoprotein VI; HER2, human epidermal growth factor receptor 2; HMGB1, high-mobility group box 1; IGF1, insulin-like growth factor 1; LXR/RXR, liver X receptor/retinoid X receptor; MSA, multiple system atrophy; NAD, nicotinamide adenine dinucleotide; Nrf2, nuclear factor-erythroid factor 2-relaed factor 2; PD-1, programmed death receptor-1; PD-L1, programmed death receptor-1 ligand; PI3K, phosphoinositide 3-kinase; PTEN, phosphatase and tensin homolog; THOP1, thimet oligopeptidase 1.

Fig. 3.. Dementia-associated proteins are associated with Alzheimer’s dementia, neuropathological changes, and CSF biomarkers.

(A) Hazard ratios (HRs) from a Cox proportional hazards model relating proteins measured in the ARIC late-life cohort to 5-year dementia risk (n = 4110; 428 cases). Late-life HRs (circles) are plotted next to midlife HRs (box) for the same protein derived from the ARIC discovery analysis. All models are adjusted for age, sex, race-study center, education, APOEϵ4 status, estimated glomerular filtration rate (eGFR) creatinine, body mass index, diabetes, hypertension, and smoking status at the time of protein assessment. (B) Beta coefficients for a cross-sectional association of candidate proteins with clinically defined Alzheimer’s disease (AD) (versus cognitively unimpaired status) and progression to AD (versus cognitively stable) among participants with mild cognitive impairment (MCI) in the EMIF-AD study derived using logistic regression. **Statistically significant (two-tailed P < 0.05) after FDR correction. *Statistically significant based on uncorrected two-tailed P < 0.05. (C) Beta coefficients for the association of candidate proteins with 20-year cognitive decline in the Whitehall II study derived using linear regression adjusted for age, sex, ethnicity, APOEϵ4 status, and eGFR. Higher values indicate elevated proteins abundance is associated with greater cognitive decline. (D) Cross-sectional association between candidate plasma proteins and CSF biomarkers in the EMIF-AD study. **Statistically significant (two-tailed P < 0.05) after FDR correction. *Statistically significant based on uncorrected two-tailed P < 0.05. Sample sizes: amyloid, n = 972; P-tau, n = 876; T-tau, n = 880; NFL, n = 643; Ng, n = 598; YKL-40, n = 649. (E) Results from a brain proteomic study of AD for candidate proteins. Results derived from Johnson et al. (18) and Wingo et al. (19). Heatmap displays signed P values. *P < 0.05; ** P < 0.01; ***P < 0.001. MCIc, mild cognitive impairment converter; MCIs, mild cognitive impairment stable; NFL, neurofilament light chain; Ng, neurogranin; p-tau181, phosphorylated tau181; T-tau, total tau; YKL-40, chitinase-3-like protein 1 (CHI3L1).

Fig. 4.. Midlife protein networks are associated with near-term and long-term dementia risk.

(A) Hierarchical cluster tree of 4877 proteins measured at baseline visit of the ARIC discovery analysis (visit 3; 1993–95). The band displays the separation of proteins into 19 modules using Netboost clustering. (B) The Topological Overlap Matrix displayed as a heatmap for visualization of protein networks. Darker green represents higher protein-protein adjacency. (C) Association of module expression (module eigenprotein) with 25-year dementia risk. (D) Association of module expression with near-term dementia risk (dementia within 15 years). (E) Association of module expression with long-term dementia risk (dementia occurring after 15 years). Hazard ratios (HRs) represent the adjusted dementia risk per standard deviation increase in module expression. (F to I) Enrichment analysis results for the proteins of modules 9, 1, 5, and 19, respectively. Top five significantly enriched pathways (P < 0.05) from each database are displayed. P value are corrected for multiple comparisons using the g:SCS algorithm in g:Profiler. Left-facing bars display pathway enrichment for proteins negatively associated with module expression. Right-facing bars display pathway enrichment for proteins positively associated with module expression. We display the six proteins in each network most highly correlated with overall network expression (hub proteins). * indicates gene encoding for hub protein is differentially expressed in AD brains as identified using the AMP-AD Sage Bionetworks Agora platform. ^★Protein has been nominated as an AD therapeutic target by AMP-AD.

Fig. 5.. Dementia-associated protein modules and enriched pathways in the decades before dementia onset.

(A) The hypothesized temporal sequence of dementia-associated protein modules and enriched biological (canonical) pathways over the 25-year follow-up period. (B) Top 50 proteins for each dementia-associated protein module based on module membership. Module membership is defined as the correlation between protein abundance and overall module expression [module eigenprotein (ME)]. Module membership values are provided in each cell.

Fig. 6.. Potential causal relationships between dementia-associated proteins and non-AD phenotypes.

Figure displays Mendelian randomization results derived from the Proteome PheWAS browser (https://epigraphdb.org/pqtl) published by Zheng et al. (27). Of the 32 dementia-associated plasma proteins identified in the present study, 9 were examined in this PheWAS study. All phenotypes displayed above were significantly associated with plasma protein level (P < 0.05) in a Mendelian-randomization analysis conducted using Wald ratio or inverse variance weighted (IVW) methods. The thickness of the arrow and associated values represents the effect size of the protein exposure on the phenotype divided by the corresponding standard error (Z-statistic). The graph displays the top six phenotypes most strongly associated with each plasma protein. The full list of phenotypes associated with each plasma protein is provided as supplementary data.