An antibody microarray analysis of serum cytokines in neurodegenerative Parkinsonian syndromes

Abstract

Background: Microarray technology may offer a new opportunity to gain insight into disease-specific global protein expression profiles. The present study was performed to apply a serum antibody microarray to screen for differentially regulated cytokines in Parkinson's disease (PD), multiple system atrophy (MSA), progressive supranuclear palsy (PSP) and corticobasal syndrome (CBS).

Results: Serum samples were obtained from patients with clinical diagnoses of PD (n = 117), MSA (n = 31) and PSP/CBS (n = 38) and 99 controls. Cytokine profiles of sera from patients and controls were analyzed with a semiquantitative human antibody array for 174 cytokines and the expression of 12 cytokines was found to be significantly altered. In a next step, results from the microarray experiment were individually validated by different immunoassays. Immunoassay validation confirmed a significant increase of median PDGF-BB levels in patients with PSP/CBS, MSA and PD and a decrease of median prolactin levels in PD. However, neither PDGF-BB nor prolactin were specific biomarkers to discriminate PSP/CBS, MSA, PD and controls.

Conclusions: In our unbiased cytokine array based screening approach and validation by a different immunoassay only two of 174 cytokines were significantly altered between patients and controls.

Figure 1

Identification of serum biomarkers for the discrimination of movement disorders by antibody arrays in the screening cohort. (A) Representative picture of Raybiotech human cytokine antibody array showing the reactivity of pooled serum samples (10 PSP/CBS, 10 MSA, 20 PD and 30 controls) to arrays G series 2000 6, 7 and 8 (174 cytokines). Each protein was measured in duplicates. Signals were scanned with a GenePix 4000B scanner. Blue boxes: positive controls (upper left corner, high intense spots). Red box, negative controls (upper left and lower right corner, no spots). Purple boxes, internal controls IC1, IC and IC3 (lower right corner, spots with three different intensities). White and green colored boxes indicate the location of the detection of two proteins that were significantly different in both the microarray and validation experiment (white = PDGF-BB and green = prolactin). (B) Normalized array data of the 174 cytokines were analyzed by SAM to detect differences in their concentrations between pooled serum samples (PSP/CBS, one pool of 10 samples with three replicates; MSA, one pool of 10 samples with three replicates each; PD, two pools of 10 samples with two replicates each; and controls, three pools of 10 samples with two replicates each). The relative concentrations of the 12 cytokines that obtained a significant score (q-value <0.001%) are shown in a “heatmap”. Low concentrations are shown in blue, median concentrations in black and high concentrations in yellow.

Figure 2

Validation of seven differentially expressed cytokines in patients with PSP/CBS, MSA and controls in the screening cohort. Individual data points are shown as circles or triangles and horizontal bars indicate medians. In addition data are shown as box plots with medians indicated as horizontal bars with boxes. Groups were compared using the Kruskal-Wallis test and Dunn’s multiple comparison post-hoc test and overall p-values for comparison of PSP/CBS, MSA and combined controls or for comparison of PSP/CBS, MSA, HC and INC are shown in each figure. Ns = statistically not significant.

Figure 3

Differential expression of PDGF-BB and prolactin in patients with PSP/CBS, MSA and controls in the screening and replication cohorts. Individual data points are shown as circles and horizontal bars indicate medians. In addition data are shown as box plot with medians indicated as horizontal bars with boxes. Groups were compared using the Kruskal-Wallis test and Dunn’s multiple comparison post-hoc test and overall p-values are shown in each figure. * Significant differences to the control group, # significant differences to PD patients.