Large-scale proximity extension assay reveals CSF midkine and DOPA decarboxylase as supportive diagnostic biomarkers for Parkinson's disease

Abstract

Background: There is a need for biomarkers to support an accurate diagnosis of Parkinson's disease (PD). Cerebrospinal fluid (CSF) has been a successful biofluid for finding neurodegenerative biomarkers, and modern highly sensitive multiplexing methods offer the possibility to perform discovery studies. Using a large-scale multiplex proximity extension assay (PEA) approach, we aimed to discover novel diagnostic protein biomarkers allowing accurate discrimination of PD from both controls and atypical Parkinsonian disorders (APD).

Methods: CSF from patients with PD, corticobasal syndrome (CBS), progressive supranuclear palsy (PSP), multiple system atrophy and controls, were analysed with Olink PEA panels. Three cohorts were used in this study, comprising 192, 88 and 36 cases, respectively. All samples were run on the Cardiovascular II, Oncology II and Metabolism PEA panels.

Results: Our analysis revealed that 26 and 39 proteins were differentially expressed in the CSF of test and validation PD cohorts, respectively, compared to controls. Among them, 6 proteins were changed in both cohorts. Midkine (MK) was increased in PD with the strongest effect size and results were validated with ELISA. Another most increased protein in PD, DOPA decarboxylase (DDC), which catalyses the decarboxylation of DOPA (L-3,4-dihydroxyphenylalanine) to dopamine, was strongly correlated with dopaminergic treatment. Moreover, Kallikrein 10 was specifically changed in APD compared with both PD and controls, but unchanged between PD and controls. Wnt inhibitory factor 1 was consistently downregulated in CBS and PSP patients in two independent cohorts.

Conclusions: Using the large-scale PEA approach, we have identified potential novel PD diagnostic biomarkers, most notably MK and DDC, in the CSF of PD patients.

Keywords: Atypical Parkinsonian disorders; Biomarker; Cerebrospinal fluid; Corticobasal syndrome; DOPA decarboxylase; Midkine; Multiple system atrophy; Parkinson’s disease; Progressive supranuclear palsy; Proximity extension assay.

Fig. 1

Altered CSF proteins between Parkinson’s disease (PD) and Controls. a Volcano plot showing log2 fold change and -log10 adjusted P-value in the Stockholm cohort (n = 69 for controls; n = 81 for PD). b Forest plot displaying all significant proteins with log fold change and 95% confidence interval ordered in ascending adjusted P-values in the Stockholm cohort. c Boxplots of the top 4 most significant proteins in the Stockholm cohort, adjusted for age and sex; levels representing normalised protein expression (NPX) and P-values displayed. T-test for groupwise comparisons (n = 69 for controls; n = 81 for PD). d Receiver operating characteristic (ROC) curves for the top 4 proteins in the Stockholm cohort with areas under curve (AUC). e Volcano plot showing log2 fold change and -log10-adjusted P-value in the BioFIND cohort (n = 37 for controls; n = 51 for PD). f Forest plot displaying all significant proteins with log fold change and 95% confidence interval ordered in ascending adjusted P-values in the BioFIND cohort. g A forest plot comparing overlapping significant proteins

Fig. 2

Association of CSF protein levels with clinical parameters of Parkinson’s disease (PD) patients. a Spearman’s rank, adjusted for age and sex, between CSF levels of significantly changed proteins in PD patients from the Stockholm cohort and disease duration, scores of Hoehn&Yahr, Unified Parkinson’s Disease Rating Scale (UPDRS) part 3, Montreal Cognitive Assessment (MoCA), Hospital Anxiety and Depression Scale (HADS), Non-Motor Symptoms Questionnaire (NMSQ) and Pittsburgh Sleep Quality Index (PSQI), and Levodopa equivalent daily dose (LEDD) (BH adjusted P-values; *P < 0.05; **P < 0.01). b Spearman’s rank, adjusted for age and sex, between CSF levels of significantly changed proteins in PD patients from the BioFIND cohort and disease duration, scores of UPDRS part 3 and MoCA, and LEDD (BH-adjusted P-values). c, d Correlation of LEDD with aromatic L-amino-acid decarboxylase levels (DDC) in the Stockholm (c) and BioFIND (d) cohorts (Spearman’s ρ, adjusted for age and sex). e Group comparisons (t-test; P-values displayed, adjusted for age and sex) between controls and PD with or without medication (PD treated and PD untreated, respectively)

Fig. 3

Comparison of CSF proteins between Parkinson’s disease (PD), atypical Parkinsonian disorders (APD) and Controls. a t-distributed Stochastic Neighbour Embedding (t-SNE) plots of PD, APD and Controls. b, c Volcano plots showing log2 fold change and -log10 of adjusted P-value for differential protein expression between APD and PD (b) and between APD and Controls (c)

Fig. 4

Comparison of CSF proteins between Parkinson’s disease (PD), atypical Parkinsonian disorders (APD) and Controls. a Boxplots of the most significantly changed proteins, adjusted for age and sex. Levels are normalised protein expression (NPX). T-test for groupwise comparisons. b Validation of Midkine (MK) levels using a commercial ELISA assay (upper) and comparison with Olink NPX values by a linear model regression (middle) and z-scored boxplot comparisons (bottom)

Fig. 5

Altered CSF proteins between 4R-Tauopathies and Controls. Volcano plots showing log2 fold change and -log10 P-value for the Stockholm (a) and the UCSF cohorts (b), with a forest plot showing that the overlapping significant proteins WIF-1 and DDC had consistent significant changes in the UCSF cohort and the Stockholm cohort (c)