Proteomic analysis of APOEε4 carriers implicates lipid metabolism, complement and lymphocyte signaling in cognitive resilience

Abstract

Background: Apolipoprotein E (APOE) ε4 allele is the strongest genetic risk factor for late onset Alzheimer's disease (AD). This case-cohort study used targeted plasma biomarkers and large-scale proteomics to examine the biological mechanisms that allow some APOEε4 carriers to maintain normal cognitive functioning in older adulthood.

Methods: APOEε4 carriers and APOEε3 homozygotes enrolled in the Women's Health Initiative Memory Study (WHIMS) from 1996 to 1999 were classified as resilient if they remained cognitively unimpaired beyond age 80, and as non-resilient if they developed cognitive impairment before or at age 80. AD pathology (Aß_42/40) and neurodegeneration (NfL, tau) biomarkers, as well as 1007 proteins (Olink) were quantified in blood collected at study enrollment (on average 14 years prior) when participants were cognitively normal. We identified plasma proteins that distinguished between resilient and non-resilient APOEε4 carriers, examined whether these associations generalized to APOEε3 homozygotes, and replicated these findings in the UK Biobank.

Results: A total of 1610 participants were included (baseline age: 71.3 [3.8 SD] years; all White; 42% APOEε4 carriers). Compared to resilient APOEε4 carriers, non-resilient APOEε4 carriers had lower Aß_42/40/tau ratio and greater NfL at baseline. Proteomic analyses identified four proteins differentially expressed between resilient and non-resilient APOEε4 carriers at an FDR-corrected P < 0.05. While one of the candidate proteins, a marker of neuronal injury (NfL), also distinguished resilient from non-resilient APOEε3 homozygotes, the other three proteins, known to be involved in lipid metabolism (ANGPTL4) and immune signaling (PTX3, NCR1), only predicted resilient vs. non-resilient status among APOEε4 carriers (protein*genotype interaction-P < 0.05). Three of these four proteins also predicted 14-year dementia risk among APOEε4 carriers in the UK Biobank validation sample (N = 9420). While the candidate proteins showed little to no association with targeted biomarkers of AD pathology, protein network and enrichment analyses suggested that natural killer (NK) cell and T lymphocyte signaling (via PKC-θ) distinguished resilient from non-resilient APOEε4 carriers.

Conclusions: We identified and replicated a plasma proteomic signature associated with cognitive resilience among APOEε4 carriers. These proteins implicate specific immune processes in the preservation of cognitive status despite elevated genetic risk for AD. Future studies in diverse cohorts will be needed to assess the generalizability of these results.

Keywords: APOE, Resilience; Alzheimer’s disease; Cognition; Immunity; Lipids; Proteomics.

Fig. 1

Study design. All participants were cognitively normal at baseline. Targeted ADRD biomarkers (Quanterix) and plasma proteomic assays (Olink) were applied to plasma collected during each participant’s baseline visit. The primary analyses compared resilient to non-resilient APOEe4 carriers on targeted Alzheimer’s disease and related dementia plasma biomarkers and plasma proteomic measurements. Figure made with BioRender.com. Abbreviations. ADRD, Alzheimer’s disease and related dementias; WHIMS, Women’s Health Initiative Memory Study

Fig. 2

Alzheimer’s disease and neurodegeneration biomarker levels among resilient, non-resilient, and 80 + impaired participants. All results were derived from an 2 × 3 (APOE genotype x cognitive status group) ANCOVA adjusted for baseline age, recruitment region, HT treatment (HT vs. Placebo), education, kidney function (eGFR), diabetes, and high cholesterol. Full results of these analyses are presented in the Supplementary Tables. Results derived from post-hoc comparison of the non-resilient and 80 + impaired groups to the resilient (reference) group ^t Interaction between APOE genotype and resilient versus 80 + imparted was statistically significant for plasma Aß_42/40 level (interaction-P = 0.03). *Difference between group and resilient group significant at P < 0.05

Fig. 3

Difference in baseline plasma protein level between resilient and impaired participants. A. The adjusted difference between resilient and non-resilient APOEε4 groups. Two horizontal reference lines indicate P value of 0.05 and 0.01. B. Effect sizes for candidate proteins that differed in our comparison of resilient to non-resilient APOEε4 participants. Effect sizes derived from APOEε4 participants are displayed on the x-axis, whereas effect sizes derived from APOEε3 participants are displayed on the y-axis. * Indicates a statistically significant interaction by APOE genotype. C. Examination of dose response effects for proteins that were (i) differentially expressed between resilient and non-resilient APOEε4 participants and (ii) showed statistically significant interaction by APOE genotype. Dose response was determined by deriving the difference between resilient and non-resilient protein levels among APOEε3/ε3, APOEε3/ε4, and APOEε4/ε4 participants. D. The adjusted difference between resilient and non-resilient APOEε3 groups. Two horizontal reference lines indicate p value of 0.05 and 0.01. E. Effect sizes for candidate proteins that differed in our comparison of resilient to non-resilient APOEε3 participants. Effect sizes derived from APOEε4 participants are displayed on the x-axis, whereas effect sizes derived from APOEε3 participants are displayed on the y-axis. * Indicates a statistically significant interaction by APOEε4 genotype. All results were derived from an 2 × 3 ANCOVA adjusted for baseline age, recruitment region, HT treatment (HT vs. Placebo), education, kidney function (eGFR), diabetes, and high cholesterol

Fig. 4

Association of candidate proteins with incident all-cause, Alzheimer’s, and vascular dementia in the UK Biobank. A. Association of APOEε4 candidate proteins with incident dementia in the UK Biobank. B. Association of APOEε3 candidate proteins with incident dementia in the UK Biobank. Hazard ratios were derived from Cox proportional hazards models adjusted for age, sex, education study site, BMI, kidney function (eGFR), diabetes, and cholesterol. Pink circles represent statistical significance at an unadjusted P < 0.05. A bolded protein name indicates significant effect modification by APOEε4 genotype in the WHIMS discovery analyses

Fig. 5

Association of resiliency-associated proteins with plasma ADRD biomarkers A. Age-adjusted correlation between plasma protein level and plasma Aß_42/40, tau, NfL, IL6 and Aß_42/42 to tau ratio for APOEe4 candidate proteins. B. Age-adjusted correlation between plasma protein level and plasma Aß_42/40, tau NfL, IL6 and Aß_42/42 to tau ratio for APOEe3 candidate proteins. The heatmaps display the results in the full sample, among APOEe4 carriers, and among APOEe3 carriers. Bolded protein name indicates significant effect modification by APOE genotype in the discovery (WHIMS) analyses

Fig. 6

Functional characterization of APOEε4 resiliency-associated proteins. A. GTEx database gene expression in brain, whole blood, and other tissues using data available from postmortem samples [57]. The expression of cognate genes coding for proteins associated with non-resilient versus resilient status are displayed in transcripts per million. Genes and tissues are grouped on the y- and x-axis based using hierarchical clustering. B. Brain and vascular cell-specific expression of genes coding for proteins associated with non-resilient versus resilient status [58]. Values are expressed in terms of average normalized counts. https://twc-stanford.shinyapps.io/human_bbb/. C-E. Brain and vascular cell-specific expression of genes coding for proteins associated with non-resilient versus resilient status. Expression values are presented stratified by Alzheimer’s disease verses control status [58]. F. Using Ingenuity Pathway Analysis (IPA), we identified molecules downstream of top candidate proteins associated with resilient versus non-resilient status among APOEe4 carriers. Relationships between molecules (edges) were defined based on activation, causation, chemical-chemical interactions, chemical-protein interactions, inhibition, modification, molecular cleavage, phosphorylation, protein-DNA interactions, protein-protein interactions, protein-RNA interactions, regulation of binding, RNA-RNA interactions, transcription, translocation, and ubiquitination. We identified the top three canonical pathways (based on number of overlapping molecules) among the candidate proteins and downstream molecules (left) and included three canonical pathways implicated in Alzheimer’s disease and related dementia (right). Bolded molecules are downstream of one or more candidate protein and implicated in AD. Abbreviations: aaSMC, Arteriolar Smooth Muscle Cell; ART, Arterial; aSMC, Vascular Smooth Muscle Cell; AST-Ctx, Astrocyte-Cortex; AST-Hpc, Astrocyte-hippocampus; CAP, Capillary; EPEN, Ependymal; M.FB, Meningeal Fibroblast; MCR, Motoric Cognitive Risk; MG, Microglia; M-PC, ECM-regulating Pericyte; NEU, Neuron; OL, Oligodendrocyte; OPC, Oligodendrocyte Precursor Cell; P.FB, Perivascular Fibroblast; PM, Perivascular Macrophage; TC, T-cell; T-PC, Solute transport-Pericyte; VEN, Venous; VINE, Vessel Isolation and Nuclei Extraction for Sequencing