CSF proteome profiling across the Alzheimer's disease spectrum reflects the multifactorial nature of the disease and identifies specific biomarker panels

Abstract

Development of disease-modifying therapies against Alzheimer's disease (AD) requires biomarkers reflecting the diverse pathological pathways specific for AD. We measured 665 proteins in 797 cerebrospinal fluid (CSF) samples from patients with mild cognitive impairment with abnormal amyloid (MCI(Aβ+): n = 50), AD-dementia (n = 230), non-AD dementias (n = 322) and cognitively unimpaired controls (n = 195) using proximity ligation-based immunoassays. Here we identified >100 CSF proteins dysregulated in MCI(Aβ+) or AD compared to controls or non-AD dementias. Proteins dysregulated in MCI(Aβ+) were primarily related to protein catabolism, energy metabolism and oxidative stress, whereas those specifically dysregulated in AD dementia were related to cell remodeling, vascular function and immune system. Classification modeling unveiled biomarker panels discriminating clinical groups with high accuracies (area under the curve (AUC): 0.85-0.99), which were translated into custom multiplex assays and validated in external and independent cohorts (AUC: 0.8-0.99). Overall, this study provides novel pathophysiological leads delineating the multifactorial nature of AD and potential biomarker tools for diagnostic settings or clinical trials.

Figure 1.. Study overview and differential abundance of CSF proteins across groups.

a, we included a total of 797 participants in the discovery cohort. Protein levels in CSF from cognitively unimpaired controls (white), MCI(Aβ+) (yellow), AD (red) and non-AD dementias (blue) were measured by antibody-based PEA technology. Differential CSF protein abundance as well as classification models were analyzed. The findings from the AD dementia vs control comparison were validated with the proteome data from an external cohort that employed the same technology (validation step 1, n=62²⁵). Custom multiplex PEA assays with the markers of interest were developed and validated in an independent multicenter cohort (validation step 2, n=513). b-d, Volcano plots show the CSF proteins that are differentially regulated in (b) MCI(Aβ+) (n=50) or (c) AD (n=230) vs. controls (n=195) and (d) AD vs. non-AD dementias (n=322). Each dot represents a protein. The beta coefficients (log₂ fold-change) are plotted versus q values values (−log₁₀-transformed). Proteins significantly dysregulated after adjusting for false discovery rate (FDR, q < 0.05) are colored in light blue. The name of the top 20 significant dysregulated CSF proteins and the top 5 with the strongest effect sizes are annotated. The total number of proteins that are down-regulated (left) or upregulated (right) is indicated. Horizontal dotted line indicates the significance threshold. CON, cognitively unimpaired controls; MCI(Aβ+), mild cognitive impairment with Aβ pathological changes; AD, Alzheimer’s disease; non-AD dem: non-AD dementias. Some images within figure 1a are courtesy of Olink Proteomics AB.

Figure 2.. Differentially regulated CSF proteins change along AD spectrum and reflect different biological processes

a) Venn diagram depicting the overlap between proteins dysregulated between MCI and controls as well as between AD and controls and non-AD dementias. Markers that were not differentially regulated between MCI or AD and controls are not included. This resulted into four specific protein groups: markers changed specifically in AD (red), markers changed in both MCI(Aβ+) and AD (orange), markers changed only in MCI(Aβ+) but not in AD (yellow), and markers changed in MCI(Aβ+) and AD or AD only, but not between AD and non-AD dementias (grey). The total number of proteins and the name of the top 5 dysregulated proteins in each of the groups is annotated b) Protein trajectories of each specific grouped defined within the Venn diagram. For each individual protein, the log₂ fold change was calculated by subtracting the mean NPX values (protein levels) of the control group from the mean NPX values in each diagnostic group. Lines connect the fold changes of each individual protein along AD disease stage. Dots represent the median and interquartile range of the fold changes for each specific clinical group. Dotted black line represent a 0-fold change. c) Bar graphs depicting the biological pathways enriched in each of the protein groups. Functional enrichment was performed using Metascape selecting GO Biological Processes as ontology source. Terms with a p-value < 0.01, a minimum count of 3, and an enrichment factor > 1.5 were collected and grouped into clusters based on their membership similarities. p-values were calculated based on the accumulative hypergeometric distribution. Kappa scores are used as the similarity metric when performing hierarchical clustering on the enriched terms, and sub-trees with a similarity of > 0.3 are considered a cluster. The most statistically significant term within a cluster is chosen to represent the cluster. The corresponding GO number and biological process is defined in the right side. Stronger colours represent higher significant enrichment. Vertical line represents the significant threshold (unadjusted p <0.01). Stronger colors represent higher significant enrichment. MCI(Aβ+), mild cognitive impairment with Aβ pathological changes; AD, Alzheimer’s disease; non-AD dem, non-AD dementias; CON, cognitively unimpaired controls

Figure 3.. CSF biomarker panels for early and specific diagnosis of AD.

Receiver operating characteristic (ROC) curves depicting the performance of CSF biomarker panels discriminating AD (a, orange) from controls in the discovery (CON = 195, AD = 230) and validation 1 (CON=44, AD=18) cohorts. b) ROC curve analyses depicting the performance of CSF biomarker panels discriminating AD (n=230) from non-AD dementias (blue, n=322) in the discovery cohort. Black line is the mean area under the curve (AUC) over all re-samplings (1000 repeats of 5-fold cross-validation, grey lines). Inserts outline corresponding AUC and 95% CI c) Correlation matrix heatmap representing the Spearman’s correlation coefficient in-between the proteins selected in each panel, the classical AD CSF biomarkers and ratios and MMSE score within the complete discovery cohort. d) Overview of all mean AUCs of each panel for the discrimination of the different groups of interest in the discovery CSF proteome profiling (CON=195, MCI(Aβ+) = 50, AD = 230, non-AD dementia = 322) and with the custom assays in validation step 2 (CON=110, MCI(Aβ+) = 101, AD = 88, non-AD dementias = 214). Error bars represent 95% CI. e) Scatter plots show the correlation between the beta-coefficients obtained in the discovery phase to those obtained with the custom assays in validation step 2. Insert indicate the spearman correlation coefficient. f) ROC curves depicting the performance of CSF biomarker panel discriminating AD (n=88) from controls (n=110) or non-AD dementias (n=214) using the custom assays. Inserts outline corresponding AUC and 95% CI. *ABL1 and ITGB2 are proteins that are present in both CSF panels. MCI(Aβ+), mild cognitive impairment with Aβ pathological changes; AD, Alzheimer’s disease; non-AD dem: non-AD dementias; CON, cognitively unimpaired controls.