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. 2019 Jul 8;42(7):zsz079.
doi: 10.1093/sleep/zsz079.

Extracellular vesicles reveal abnormalities in neuronal iron metabolism in restless legs syndrome

Sahil Chawla  1 , Seema Gulyani  2 , Richard P Allen  3 , Christopher J Earley  3 , Xu Li  4 , Peter Van Zijl  4 , Dimitrios Kapogiannis  1
Affiliations

Extracellular vesicles reveal abnormalities in neuronal iron metabolism in restless legs syndrome

Sahil Chawla et al. Sleep. .

Abstract

Study objectives: Determine abnormalities in levels of iron-management proteins in neuronal origin-enriched extracellular vesicles (nEVs) in restless legs syndrome (RLS).

Methods: We used immunoprecipitation for neuronal marker L1CAM to isolate nEVs from the serum of 20 participants with RLS from a study including magnetic resonance imaging (MRI) determinations of iron deposition in the substantia nigra and hematologic parameters and 28 age- and sex-matched Controls.

Results: RLS compared with Control participants showed higher levels of nEV total ferritin but similar levels of transferrin receptor and ferroportin. Western blot analysis showed that heavy- but not light-chain ferritin was increased in nEVs of RLS compared with Control participants. In RLS but not Control participants, nEV total ferritin was positively correlated with systemic iron parameters; the two groups also differed in the relation of nEV total ferritin to MRI measures of iron deposition in substantia nigra.

Conclusions: Given the neuronal origin and diversity of EV cargo, nEVs provide an important platform for exploring the underlying pathophysiology and possible biomarkers of RLS.

Keywords: exosomes; extracellular vesicles; ferritin; iron; restless legs syndrome.

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Figures

Figure 1.
Figure 1.
Iron management proteins in L1CAM + nEVs. Graphs depict values (mean ± SD) of Transferrin Receptor (TfR) (A), Ferroportin (B), and Ferritin (C) in 20 RLS and 18 Control participants—except for 17 and 28 Control participants for Ferritin (C) (ns = nonsignificant). In (C) * depicts p = 0.032 in the first graph (including RLS and Control participants from JH) and p = 0.017 in the second graph (including additional Control participants from the NIA); values below the LLoQ with CV < 15% were assigned the LLoQ value. Group differences remained significant, even if we analyzed raw signal or did not set concentration values to the LLoQ.
Figure 2.
Figure 2.
Altered Ferritin profile in L1CAM + nEVs in RLS. (A, B) People with RLS with the highest ferritin concentration and Control participants with the lowest ferritin concentrations were selected. Western blot (WB) image shows heavy-chain ferritin (FTH), light-chain ferritin (FTL), and Alix, a common nEV marker used for normalization. Normalized FTH was higher (** depicts p = 0.009) in people with RLS compared with Controls; there were no differences in FTL (ns refers to p = 0.17). (C, D) People with RLS and Controls with equivalent ferritin concentrations were selected. Normalized FTH was again higher (* depicts p = 0.04) in people with RLS compared with Controls; again there were no differences for FTL (ns refers to p = 0.45). Graphs depict mean ± SEM.
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