Prior to this study, only specific iron-related proteins and dopaminergic metabolites have been examined in the CSF of RLS/WED patients [15, 16]. Although the importance of iron in the etiology of early-onset RLS/WED has been established, our proteomic approach in this study permitted identification of proteins that could prove valuable in understanding pathways affected in RLS/WED and identifying targets for treatment strategies.
In this discovery study, we have utilized a 2D-DIGE coupled with tandem MS to identify 6 candidate CSF protein markers for early-onset Restless Legs Syndrome (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 compared to controls.
Cystatin C levels were increased in the CSF of early-onset RLS/WED patients. Cystatin C is a cysteine proteinase inhibitor that has multiple functions in vivo. It is known to inhibit both endogenous and exogenous cysteine peptidases, regulate the immune system, have antibacterial and/or antiviral effects; and neuroprotective effects during brain injury. Cystatin C has been described previously as a biomarker of pain in lumbar disk herniation, sciatica, and labor [17, 18]. The level of cystatin C observed in the CSF of patients with these conditions was found to be dependent on the pain’s intensity and duration. The increased expression of cystatin C in the CSF from RLS/WED patients is consistent with multiple reports of pain associated with RLS/WED symptoms and increased temporal summation to heat pulses in RLS/WED patients suggesting that RLS/WED is associated with amplified CNS pain processing [19, 20]. Also, RLS/WED has significant comorbitity with a number of pain conditions such as fibromyalgia  and headache . The finding of elevated cystatin C in the CSF of RLS/WED subjects may provide an objective measure of the pain associated with RLS/WED in some of the patients. Also cystatin C has been reported to be elevated in response to inflammation; therefore, there is the potential that immune system activation may be occurring in the CNS of patients with RLS/WED. A recent review by Weinstock et al.  suggests that some conditions that are associated with RLS/WED have an inflammatory/immune component and that inflammation may be responsible for the CNS iron deficiency.
In this study, we found increased levels of lipocalin-type prostaglandin D2 synthase (L-PGDS) in RLS/WED CSF. L-PGDS is the most abundant protein in the CSF and serves two roles: 1) synthesis of prostaglandin D2 (PGD2) from prostaglandin H2 (PGH2); and 2) binding and transporting small lipophilic molecules. L-PGDS alterations have been reported in a number of sleep disorders including narcolepsy and obstructive sleep apnea with and without daytime sleepiness [24–26] and thus its increase is relevant to sleep disruptions in RLS/WED. We have reported that PGDS is decreased in CSF of monkeys that were iron deficient . Furthermore, the increase in PGDS in RLS/WED CSF is inconsistent with the reports of decreased brain iron status in RLS/WED . We suggest that the increase in PGDS that was observed in RLS/WED patients could be part of a compensatory mechanism. It has been reported that L-PGDS is upregulated in the brains of subjects with hypoxic-ischemic encephalopathy (HIE) . Also, it has been demonstrated in a mouse model of HIE that L-PGDS and HIF-1α expression are co-localized and it was implied that L-PGDS may protect neurons from hypoxic insult by local secretion of PGD2. We could hypothesize, therefore, that the increased L-PGDS in RLS/WED may be a result of hypoxia pathway activation . This hypothesis is further supported by our recent finding of HIF pathway activation in the brains and microvasculature of RLS/WED subjects .
In this study, we report elevated vitamin D binding protein in the CSF of RLS/WED subjects. Vitamin D binding protein (VDP) is noted for specifically binding and transporting a number of sterols including vitamin D metabolites to target cells, however, it has been also been shown to be a scavenger of toxins that are released due to cellular injury or death  and a modulator of the immune response . Alterations in cerebrospinal fluid levels of VDP have been linked primarily to neurodegenerative diseases, such as multiple sclerosis, Parkinson’s disease, and trauma [17, 30, 31]. Vitamin D also has important ties to the pathways that have been identified in the etiology of RLS/WED. Vitamin D deficiency has been linked to altered dopamine concentrations in the cortex , and blood and tissue iron content has been reported to increase following vitamin D supplementation . Balaban et al. have reported decreased serum 25-hydroxyvitamin D levels in female RLS/WED subject and have postulated that the CNS iron deficiency that occurs in RLS/WED may induce alterations in the dopaminergic pathway via a vitamin D-mediated pathway . Because of the tie between iron, dopamine and vitamin D, it is tempting to propose that elevated VDP that we observed in this study could be reflective of the alterations in the iron/dopamine pathway. One could also hypothesize that the elevation of VDP could also be reflective of activation of inflammatory/immune cascades in the CNS of RLS/WED subjects as further supported by the alterations of cystatin C and alpha-1 acid glycoprotein levels in RLS/WED CSF.
Beta-Hemoglobin was found to be increased in RLS/WED CSF. β-Hemoglobin is a major component of adult hemoglobin and normally is not present in the CSF. As such, the presence of β-Hemoglobin in the CSF is often considered to be indicative of blood contamination. While, obtaining a CSF tap with no blood contamination is extremely difficult, we have examined the CSF for frank signs of blood to rule out contamination. β-Hemoglobin is known to transport oxygen in the blood, however, it has also been reported to be important in dopamine regulation in the brain . Biagioli and colleagues have reported that overexpression of Hbb-b1 and Hba-a1 (the genes encoding Hemoglobin β and Hemoglobin α) in dopaminergic cell lines alters transcription of genes related to iron metabolism, oxygen homeostasis, oxidative phosphorylation, and nitric oxide synthesis . Because evidence exists for the disruption of both the dopaminergic and oxygen homeostatic systems in RLS [27, 36–38], it is tempting to suggest the finding of increased β-hemoglobin in the RLS/WED CSF may reflect a more chronic condition in RLS/WED and perhaps may point to a mechanistic explanation for the altered iron, dopamine and oxygen protein profiles that are observed in RLS/WED.
Apolipoprotein A1 was found to be decreased in the CSF of patients with RLS/WED. Apolipoprotein A1 is the primary protein constituent of high density lipoproteins (HDL). Although the role of Apolipoprotein A1 in the CNS is not completely understood, it is thought to be important for transport of lipids into the brain . To date, no association has been reported between Apo-A1 and RLS/WED, however, recent studies [40, 41] have described a higher prevalence of hypercholesterolemia in RLS/WED subjects. Decreased serum Apo-A1 levels have been linked to increased incidence of cardiovascular disease  which, if the decrease in Apo-A1 that was observed in RLS/WED CSF was reflective of a global Apo-A1 level, this could be related to the increased risk for cardiovascular disease in RLS/WED [4–7].
Alpha-1-acid glycoprotein levels were decreased in the CSF from RLS/WED patients. Alpha-1-acid glycoprotein (AGP) is synthesized primarily in the liver, but also is reportedly synthesized in the endothelial cells of the blood–brain-barrier. Alpha-1-acid glycoprotein (AGP) is an acute phase reactant that is involved in the inflammation cascade and is thought to provide immediate and effective response to inflammatory damage prior to successive protective mechanisms . Their protective functions seem to result mainly from iron sequestration [44, 45]. Our finding of decreased AGP in the CSF of RLS/WED subjects is reflective of decreased iron content in the CNS and provides further support for altered iron homeostasis in the brains of RLS/WED patients [14, 46, 47].