Alzheimer disease pathology and the cerebrospinal fluid proteome

Abstract

Background: Altered proteome profiles have been reported in both postmortem brain tissues and body fluids of subjects with Alzheimer disease (AD), but their broad relationships with AD pathology, amyloid pathology, and tau-related neurodegeneration have not yet been fully explored. Using a robust automated MS-based proteomic biomarker discovery workflow, we measured cerebrospinal fluid (CSF) proteomes to explore their association with well-established markers of core AD pathology.

Methods: Cross-sectional analysis was performed on CSF collected from 120 older community-dwelling adults with normal (n = 48) or impaired cognition (n = 72). LC-MS quantified hundreds of proteins in the CSF. CSF concentrations of β-amyloid 1-42 (Aβ_1-42), tau, and tau phosphorylated at threonine 181 (P-tau181) were determined with immunoassays. First, we explored proteins relevant to biomarker-defined AD. Then, correlation analysis of CSF proteins with CSF markers of amyloid pathology, neuronal injury, and tau hyperphosphorylation (i.e., Aβ_1-42, tau, P-tau181) was performed using Pearson's correlation coefficient and Bonferroni correction for multiple comparisons.

Results: We quantified 790 proteins in CSF samples with MS. Four CSF proteins showed an association with CSF Aβ_1-42 levels (p value ≤ 0.05 with correlation coefficient (R) ≥ 0.38). We identified 50 additional CSF proteins associated with CSF tau and 46 proteins associated with CSF P-tau181 (p value ≤ 0.05 with R ≥ 0.37). The majority of those proteins that showed such associations were brain-enriched proteins. Gene Ontology annotation revealed an enrichment for synaptic proteins and proteins originating from reelin-producing cells and the myelin sheath.

Conclusions: We used an MS-based proteomic workflow to profile the CSF proteome in relation to cerebral AD pathology. We report strong evidence of previously reported CSF proteins and several novel CSF proteins specifically associated with amyloid pathology or neuronal injury and tau hyperphosphorylation.

Keywords: Alzheimer disease; Amyloid; Biomarker; CSF; Cerebrospinal fluid; Mass spectrometry; Proteomics; Tandem mass tag; Tau.

Fig. 1

Study design and cerebrospinal fluid (CSF) proteome profiling workflow. CSF samples from 120 older individuals with or without cognitive impairment were analyzed using a highly automated shotgun MS-based proteomic workflow. The workflow consists of first removing 14 highly abundant proteins in CSF by immunoaffinity. The rest of the workflow is automated in a 96-well plate format and includes steps of (1) reduction, alkylation, and enzymatic digestion; (2) isobaric labeling and pooling; and (3) purifications. The samples are analyzed with reversed-phase LC-MS/MS, and the data are processed with standard bioinformatic tools

Fig. 2

Cerebrospinal fluid (CSF) proteins relevant to Alzheimer pathology. Box plots of CSF proteins according to CSF tau phosphorylated at threonine 181 (P-tau181)/β-amyloid 1–42 (Aβ_1–42) ratio (i.e., “high” when P-tau181/Aβ_1–42 > 0.0779 [blue dots] and “low” when P-tau181/Aβ_1–42 ≤ 0.0779 [red dots]) for positive and negative CSF profiles of AD pathology, respectively, in all subjects (a) and restricted to subjects with cognitive impairment (b). In total, 541 CSF proteins were tested (one by one) in a logistic regression model. P values were corrected for multiple testing using the Benjamini-Hochberg procedure. Box plots were produced for the significant hits presenting false discovery rate ≤ 5%. Relative protein fold change ratios were used (in Log2). Human proteins in the box plots are given by their UniProtKB/Swiss-Prot entry name

Fig. 3

Correlations of cerebrospinal fluid (CSF) proteins with β-amyloid 1–42 (Aβ_1–42), tau, and tau phosphorylated at threonine 181 (P-tau181) concentrations in CSF. Correlation of CSF proteins with CSF Aβ_1–42 (a), CSF tau (b), and CSF P-tau181 (c). Only significant correlations with a p value ≤ 0.05 after Bonferroni correction for multiple testing were retained and are displayed in the graphs. CSF proteins correlating with CSF Aβ_1–42, tau, and P-tau181 are illustrated in a Venn diagram (d)

Fig. 4

Annotations of cerebrospinal fluid (CSF) proteins correlating with β-amyloid 1–42 (Aβ_1–42), tau, and/or tau phosphorylated at threonine 181 (P-tau181) concentrations in CSF. Tissue annotation using the UniProt tissue annotation database (a) and Gene Ontology (GO) (cellular component category) annotation (b) obtained with DAVID software for the 59 CSF proteins correlating with CSF Aβ_1–42, tau, and/or P-tau181. Significant enrichment (Benjamini-Hochberg procedure) is indicated with an asterisk. The background used for the enrichment analysis was the 790 detected proteins in CSF. n.s. Nonsignificant