Impact of iron-deficiency upon behaviour and protein expression in the male mouse with implications for Restless Legs Syndrome

Abstract

Restless legs syndrome (RLS) comprises of both sensory and motor symptoms, observed as an increased pain and periodic jerking movement of the legs. There are two forms of RLS; one being the genetic (primary) form whereby 4 gene variants have been identified, the second form is non-genetically characterised (secondary), where iron-deficiency is of major importance. Dopaminergic therapy has a central role in treating both forms of RLS suggesting an important function of the dopaminergic system, which in turn is known to be dysregulated by iron-deficiency via iron dependence of tyrosine hydroxylase and the dopamine transporter. The aim of this thesis was to devise an animal model of secondary RLS and to correlate whether iron-deficiency is the inducing factor of the behavioural sensory and motor symptoms in-vivo.A nutritional deprivation of iron in C57Bl/6 wildtype (WT) and dopamine receptor 3 null (D3-/-) male mice was induced by an iron-deficient (ID) diet starting from post-natal day 28 (P28) and continuing for 1, 4, 8, 15 or 30 weeks duration. By using the hot-plate and formalin tests after 1, 4 and/or 15 weeks of ID diet, acute and persistent sensory responses were tested upon activation of primarily afferent Aδ- and C-fibres, respectively, where these fibres have been shown in clinical studies to be altered in both primary and secondary RLS patients. c-Fos immunoreactive (c-Fos-IR) cells at the ipsilateral horn were quantified accordingly. A voluntary wheel-running system measured changes in circadian running activity after 4 and 15 weeks of ID diet. Protein analysis was performed on the entire midbrain region from the 15 week ID and control WT mouse groups. Screening for proteins that show changes during iron-deficiency was performed by two dimension fluorescence difference in gel electrophoresis (2-D DIGE). Afterwards, proteins were identified using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) followed by quantitative Western blotting and 2-D isoelectic focussing followed by SDS-PAGE (2-D IEF/SDS-PAGE).1) The acute pain response was elevated in ID WT mice after 4 and 15 weeks. After 15 weeks of diet, D3-/- mice exhibit an increased acute pain response compared to WT mice. Iron-deficiency did not further heighten the acute pain response in D3-/- mice.2) The persistent pain response was increased in ID WT mice after all durations of ID diet (1, 4 and/or 15 weeks). After 15 weeks of diet, D3-/- mice showed an elevated pain response which was further increased by iron-deficiency.3) After 15 weeks of ID diet, an increased running activity (number of runs and activity time) was shown in ID WT mice specifically 2 hours before the end of the active dark phase (06:00hr-07:00hr). ID D3-/- mice showed a heightened running activity at an earlier time period (03:00hr-04:00hr).4) In total 14 protein targets were dysregulated, 5 of which were identified by MALDI-TOF-MS. One of these proteins was sirtuin 2 (SIRT2), where its expression was elevated by 20% in ID WT mice. Quantitative Western blotting and 2-D IEF/SDS-PAGE identified non-significant changes, however all techniques confirm a similar increase in SIRT2 protein expression at the short SIRT2 variant 2 (v2) localised predominantly in myelin. Isoform 2 of SIRT v2 which has an unknown function shows this increase. Proteolipid protein/DM20 (PLP/DM20) and dopamine transporter (DAT) show similar trends to that previously described in other ID rodent studies.The work reported in this thesis has identified iron-deficiency as a factor in increasing both the sensory perception and modifying the circadian onset of increased motor activity. The pain tests show increased acute and persistent inflammatory pain responses, in addition to an elevated motor activity and activity time prior to the sleep phase in mice, corresponding to a period showing increased RLS complaints in humans. In the absence of D3 receptors, iron-deficiency has a synergistic role in shifting the onset of heightened activity to an earlier onset by at least 2 hours, which is characteristic of augmentation described in human RLS under dopaminergic therapy. These behavioural tests have proved effective in describing both sensory and motor changes which are comparable to those in RLS patients. Moreover, this is the first animal model which shows the closest match to RLS symptoms. In addition to the behavioural studies, the identification of SIRT2 in relation to iron-deficiency by using MALDI-TOF-MS is a novel finding. To date there is no animal model based on the genetic variants identified. Therefore, investigating changes caused by iron-deficiency in the mouse compared to the rat due to its fully identified genome, offers the most promising approach. Findings from these mouse studies will prove important in the future by contributing to the design of a genetic mouse model of RLS.