Cerebrospinal Fluid from Restless Legs Syndrome Patients Reduces Iron Uptake in Blood–Brain Barrier Endothelial Cells by Disrupting the Regulation of Transferrin Receptors

Objective Restless legs syndrome (RLS) is a sensorimotor disorder marked by an uncontrollable urge to move the legs. A pathophysiological hallmark of RLS is brain iron deficiency. The endothelial cells (ECs) of the blood–brain barrier (BBB) are responsible for regulating brain iron uptake. Our objective is to determine if brain iron uptake is altered in ECs in RLS. Methods Human ECs were generated from induced pluripotent stem cells (iPSCs). ECs were exposed to RLS (n = 14) or control cerebrospinal fluid (CSF) (n = 15), and 57 Fe‐Transferrin transport was determined. Immunoblotting and quantitative polymerase chain reaction were used to analyze protein, microRNA (miRNA), and messenger RNA (mRNA) expressions. We performed miRNA and transferrin receptor 1 (TfR1) 3′ iron‐responsive elements (IRE) interaction in HEK‐293 cells by using a pMIR‐REPORTER luciferase vector. Results Free and protein‐bound iron in CSF from RLS subjects were decreased compared to controls. Exposure of ECs to RLS CSF significantly decreased the uptake and transport of 57 Fe compared to the control. TfR1 expression decreased while iron regulatory proteins (IRP) increased in RLS‐CSF exposed ECs. TfR1 expression was also regulated by miRNAs. The miR‐124‐3p levels were higher in RLS CSF‐derived extracellular vesicles than in the control. It binds the TfR1 3′ IRE sequence in the pMIR‐REPORTER vector, reducing luciferase expression. Interpretation When ECs are treated with CSF from RLS patients, they show a profile of iron deficiency, except that the TfR1 expression does not increase as would be predicted. The decrease in TfR1 protein expression is because of a reduction in TfR1 mRNA stability by the binding of increased miR‐124‐3p. ANN NEUROL 2026;100:48–58