Proteomic analysis of the cerebrospinal fluid of patients with restless legs syndrome/Willis-Ekbom disease

Abstract

Background: Restless Legs Syndrome/Willis-Ekbom Disease (RLS/WED) is a sensorimotor disorder that causes patients to experience overwhelming and distressing sensations in the legs compelling the patient to move their legs to provide relief. The purpose of this study was to determine if biomarkers in the cerebrospinal fluid can distinguish RLS/WED patients from neurological controls.

Methods: We obtained CSF samples by lumbar puncture from 5 early-onset RLS/WED patients and 5 controls. We performed 2-dimensional difference in-gel electrophoresis (2D-DIGE). Proteins that were significantly altered were identified by Student's t-test. Protein spots that were differentially expressed (p ≤ 0.05, Av. Ratio ≥ 2.0) between RLS/WED and control CSF samples were identified using MALDI-TOF-MS. Statistical analyses of the validation immunoblot assays were performed using Student's t-test.

Results: In this discovery study we identified 6 candidate CSF protein markers for early-onset RLS/WED. Four proteins (Cystatin C, Lipocalin-type Prostaglandin D2 Synthase, Vitamin D binding Protein, and β-Hemoglobin) were increased and 2 proteins (Apolipoprotein A1 and α-1-acid Glycoprotein) were decreased in RLS/WED patients.

Conclusions: Our results reveal a protein profile in the RLS/WED CSF that is consistent with clinical findings of disruptive sleep, cardiovascular dysfunction and painful symptoms. Moreover, protein profiles are consistent with neuropathological findings of activation of hypoxia inducible factor (HIF) pathways and alterations in dopaminergic systems. These data indicate the CSF of RLS/WED patients may provide information relevant to biological basis for RLS/WED, treatment strategies and potential new treatment targets.

Figure 1

Representative 2D-DIGE analysis of RLS/WED versus Control CSF. This figure represents a single 2D-DIGE analysis performed on the CSF from RLS/WED and Control subjects. Differentially expressed proteins are encircled in red. Differentially expressed proteins that were able to be identified by MS/MS are listed in the legend.

Figure 2

Immunoblot quantification for cystatin C in CSF of RLS/WED and control subjects. This figure demonstrates that RLS/WED CSF contains higher Cystatin C levels compared to control subjects. This data is compiled from the CSF from 5 RLS/WED and 5 control patients and each sample was analyzed in triplicate. Single line reflects the median value and the asterisk (*) reflect statistical significance (p <0.05) as determined by an unpaired t-test analysis.