Elevated levels of cerebrospinal fluid α-synuclein oligomers in healthy asymptomatic LRRK2 mutation carriers

Abstract

Mutations in the leucine-rich repeat kinase 2 gene are the most common cause of autosomal dominant Parkinson's disease (PD). To assess the cerebrospinal fluid (CSF) levels of α-synuclein oligomers in symptomatic and asymptomatic leucine-rich repeat kinase 2 mutation carriers, we used enzyme-linked immunosorbent assays (ELISA) to investigate total and oligomeric forms of α-synuclein in CSF samples. The CSF samples were collected from 33 Norwegian individuals with leucine-rich repeat kinase 2 mutations: 13 patients were clinically diagnosed with PD and 20 patients were healthy, asymptomatic leucine-rich repeat kinase 2 mutation carriers. We also included 35 patients with sporadic PD (sPD) and 42 age-matched healthy controls. Levels of CSF α-synuclein oligomers were significantly elevated in healthy asymptomatic individuals carrying leucine-rich repeat kinase 2 mutations (n = 20; P < 0.0079) and in sPD group (n = 35; P < 0.003) relative to healthy controls. Increased α-synuclein oligomers in asymptomatic leucine-rich repeat kinase 2 mutation carriers showed a sensitivity of 63.0% and a specificity of 74.0%, with an area under the curve of 0.66, and a sensitivity of 65.0% and a specificity of 83.0%, with an area under the curve of 0.74 for sPD cases. An inverse correlation between CSF levels of α- synuclein oligomers and disease severity and duration was observed. Our study suggests that quantification of α-synuclein oligomers in CSF has potential value as a tool for PD diagnosis and presymptomatic screening of high-risk individuals.

Keywords: CSF; LRRK2 mutation carriers; Parkinson’s disease; alpha-synuclien; biomarkers.

Figure 1

Levels of t-α-syn (in ng/ml) in CSF from healthy controls (HC), sporadic PD patients (sPD), symptomatic PD patients with LRRK2 mutations (LRRK2-PD), and healthy asymptomatic LRRK2 mutation carriers (LRRK2-H). Values are expressed as the mean ± standard deviation.

Figure 2

(A) Size exclusion chromatographic analysis of α-syn oligomers in CSF. Five mls of CSF from a PD patient concentrated to 0.5 ml, was fractionated on a superdex 200 SE column. The elution volume for monomeric (M) α-syn was determined by fresh recombinant α-syn solution and molecular weight standard, and was eluted in a peak corresponding to column volume of 13–14 ml (kDa ~68), while dimeric (D) α-syn was eluted in a peak corresponding to column volume of approximately 12 ml (kDa ~170). High molecular weight oligomeric α-syn was determined by aged recombinant α-syn solution and molecular weight standard, and was eluted in a peak corresponding to column volume of approximately 9 ml (670 kDa). The void volume (V) corresponds to MW >670 kDa. The fractions of 1 ml which correspond to the oligomeric and monomeric α-syn peaks were separately collected, concentrated to 100 μl using a speed vac, and then analyzed by western blotting for the presence of α-syn using anti-α-syn (211) antibody (1:1000). (B) Scatter plot representing individual values of the levels of o-α-syn (in relative luminescence units/second [RLU/s]) in CSF from healthy controls (HC; open circles), symptomatic PD patients with LRRK2 point mutations (LRRK2-PD; open triangles), patients with (sPD; inverted open triangles), and healthy asymptomatic LRRK2 mutation carriers (LRRK2-H; open squares). Each bar represents the mean value.

Figure 3

Scatter plot presenting individual values for the ratio of o-α-syn to t-α-syn (o-α-syn/t-α-syn ratio,%) in CSF from healthy controls (HC; open circles), symptomatic PD patients with LRRK2 point mutations (LRRK2-PD; open triangles), patients with (sPD; inverted open triangles), and healthy asymptomatic LRRK2 mutation carriers (LRRK2-H; open squares). Each bar represents the mean value. The P value for each regression line is shown in each subfigure.

Figure 4

Use of receiver operating characteristic (ROC) curves for the levels of CSF t-α-syn, o-α-syn and o-α-syn/t-α-syn ratio (%) to discriminate between (sPD) patients and controls (A), and healthy asymptomatic LRRK2 mutation carriers (LRRK2-H) patients and controls (B).