Identification of an APOE ε4-specific blood-based molecular pathway for Alzheimer's disease risk

Abstract

Introduction: The precise apolipoprotein E (APOE) ε4-specific molecular pathway(s) for Alzheimer's disease (AD) risk are unclear.

Methods: Plasma protein modules/cascades were analyzed using weighted gene co-expression network analysis (WGCNA) in the Alzheimer's Disease Neuroimaging Initiative study. Multivariable regression analyses were used to examine the associations among protein modules, AD diagnoses, cerebrospinal fluid (CSF) phosphorylated tau (p-tau), and brain glucose metabolism, stratified by APOE genotype.

Results: The Green Module was associated with AD diagnosis in APOE ε4 homozygotes. Three proteins from this module, C-reactive protein (CRP), complement C3, and complement factor H (CFH), had dose-dependent associations with CSF p-tau and cognitive impairment only in APOE ε4 homozygotes. The link among these three proteins and glucose hypometabolism was observed in brain regions of the default mode network (DMN) in APOE ε4 homozygotes. A Framingham Heart Study validation study supported the findings for AD.

Discussion: The study identifies the APOE ε4-specific CRP-C3-CFH inflammation pathway for AD, suggesting potential drug targets for the disease.Highlights: Identification of an APOE ε4 specific molecular pathway involving blood CRP, C3, and CFH for the risk of AD.CRP, C3, and CFH had dose-dependent associations with CSF p-Tau and brain glucose hypometabolism as well as with cognitive impairment only in APOE ε4 homozygotes.Targeting CRP, C3, and CFH may be protective and therapeutic for AD onset in APOE ε4 carriers.

Keywords: Alzheimer's disease; C‐reactive protein; age‐related macular degeneration; amyloid beta peptide; apolipoprotein E; cerebrospinal fluid phosphorylated tau; cognitive impairment; complement C3; complement factor H; hypometabolic convergence index; positron emission tomography.

FIGURE 1

Characterization of plasma protein clusters and their associations with AD biomarkers in an APOE genotype dependent manner. (A) WGCNA was performed using 146 plasma proteins for protein clustering, and protein modules were detected from the dendrogram using a dynamic tree‐cutting algorithm. A total of 15 modules were identified, and labeled by different colors. (B) Based on their associations with AD diagnosis (Figure S2C in supporting information) or having APOE protein, five modules were chosen for their relationships with the AD traits. Module eigengenes (MEs) and their correlations with AD biomarkers, including APOE ε4 genotype, CSF Aβ42, and p‐tau, brain HCI and ADAS were determined by using Pearson correlation in the whole sample. The correlation coefficients (with a P‐value in parentheses) are shown. Blocks were painted with different colors, representing the degrees of significance, that is, red, positive and blue, negative. (C) Stratification based on the number of APOE ε4 alleles, non‐carriers (ε4 = 0), heterozygotes (ε4 = 1), and homozygotes (ε4 = 2), was performed and the same correlation analyses as in (B) were conducted in each APOE genotype group. The Green module's associations except CSF Aβ42 were found to be APOE ɛ4 homozygote genotype dependent with statistical significance; the Black module's associations were modest in the APOE ɛ4 non‐carrier genotype. Aβ, amyloid beta; AD, Alzheimer's disease; ADAS, Alzheimer's Disease Assessment Scale‐Cognitive Subscale; APOE, apolipoprotein E; CSF, cerebrospinal fluid; HCI, hypometabolic convergence index; p‐tau, phosphorylated tau; WGCNA, weighted gene co‐expression network analysis.

FIGURE 2

The associations between plasma proteins and the AD biomarkers after stratification with APOE ε4 genotype. ADNI participants were divided into three groups based on the number of APOE ε4 alleles: non‐carriers (ε4 = 0), heterozygotes (ε4 = 1), and homozygotes (ε4 = 2). The relationships among three plasma proteins, CRP (A), C3 (B), and CFH (C), and the AD biomarkers, for example, CSF p‐tau, the HCI, and (ADAS, at baseline were examined. Scatterplots with a linear regression line with 95% confidence bands (the shaded area), the Pearson coefficient of correlations and their P‐values were used to illustrate a dose‐dependent relationships between a plasma protein (X‐axis) and an AD biomarker (Y‐axis). AD, Alzheimer's disease; ADAS, Alzheimer's Disease Assessment Scale‐Cognitive Subscale; ADNI, Alzheimer's Disease Neuroimaging Initiative; APOE, apolipoprotein E; C3, complement C3; CFH, complement factor H; CRP, C‐reactive protein; CSF, cerebrospinal fluid; HCI, hypometabolic convergence index; p‐tau, phosphorylated tau; WGCNA, weighted gene co‐expression network analysis.

FIGURE 3

The association between plasma proteins and brain glucose metabolism after stratification with APOE ε4 genotype. ADNI participants were divided into three groups based on APOE ε4 allele: non‐carriers (ε4 = 0), heterozygotes (ε4 = 1), and homozygotes (ε4 = 2). Associations of plasma CRP, C3, and CFH with FDG PET scores at different brain regions were analyzed across three APOE groups. The FDG PET brain region variables were globally normalized cerebral glucose metabolism (CMRgl). All models were partial correlations between each of the three plasma proteins (CRP, C3, CFH) and CMRgl with adjusted sex and age. Only the brain regions with P values < 0.05 were shown. We found that all plasma CRP, C3, and CFH proteins were negatively associated with FDG scores mainly in the right (R) default mode network (DMN) only in APOE ε4 homozygotes. Specifically, the brain regions included right (R) parietal inferior cortex, middle and inferior temporal cortex, R temporal pole, R supramarginal cortex, R fusiform, R insula, R and left (L) precuneus, and L cingulum (also refer to Table S2 in supporting information; for the detailed information with the Bonferroni correction). ADAS, Alzheimer's Disease Assessment Scale‐Cognitive Subscale; ADNI, Alzheimer's Disease Neuroimaging Initiative; APOE, apolipoprotein E; C3, complement C3; CFH, complement factor H; CRP, C‐reactive protein; CSF, cerebrospinal fluid; FDG, fluorodeoxyglucose; PET, positron emission tomography.

FIGURE 4

Comparison of the blood protein levels across APOE genotypes and different diagnoses. All variables (CRP, C3, CFH) underwent a log10 transformation, followed by z score rescaling to reduce skewness. After different stratifications, violin boxplots were used to depict the distribution of three blood proteins and the concentrations were compared by using Kruskal–Wallis test. (A) The comparisons were conducted among APOE ε4 non‐carriers, ε4 heterozygous, and ε4 homozygous carriers. (B) The protein levels were compared across three diagnosis groups: cognitive normal control (normal), MCI, and AD. (C) The participants were first stratified based on APOE ε4 carriers’ status and then further by the diagnoses. The comparisons across the diagnosis groups in each APOE genotype were conducted. The P values for statistical significance are shown. AD, Alzheimer's disease; APOE, apolipoprotein E; C3, complement C3; CFH, complement factor H; CRP, C‐reactive protein; MCI, mild cognitive impairment.

FIGURE 5

System biology characterization of APOE ε4–specific pathways. (A) The top pathways of a functional enrichment analysis for the 37 proteins in the five WGCNA modules (Green, Black, Turquoise, Gray, and Brown) are depicted. The numbers of proteins involved in each pathway are illustrated within each bar. (B) The proteins in the Green module were further analyzed by using the PPI network analyses by using STRING program. The extended interactions with CRP, C3, and CFH in PPI were found for the interactions with two groups of proteins outside of the Green module: (1) other proinflammatory proteins and (2) lipid metabolism proteins including APOE and other lipid proteins in the Brown module. (C) This study suggested a novel pathological pathway for AD risk in APOE ε4 genotype. During peripheral inflammation, an activated cascade, including circulating CRP, C3, and CFH, responds to external or internal infection/injury. In cerebrovasculature, this process is enhanced by APOE ε4, but suppressed by APOE ε2, leading to AD pathogenesis in the brain; in the eye, this circulating cascade affects the pathological process leading to AMD. AD, Alzheimer's disease; AMD, age‐related macular degeneration; APOE, apolipoprotein E; C3, complement C3; CFH, complement factor H; CRP, C‐reactive protein; PPI, protein–protein interaction; WGCNA, weighted gene co‐expression network analysis.