Regulation of iron movement across the blood-brain barrier

Open Access
- Author:
- Duck, Kari Ann
- Graduate Program:
- Cell and Developmental Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- August 11, 2016
- Committee Members:
- James Robert Connor, Dissertation Advisor/Co-Advisor
James Robert Connor, Committee Chair/Co-Chair
Colin James Barnstable, Committee Member
Patricia Mclaughlin, Committee Member
Ian Alexander Simpson, Outside Member - Keywords:
- Blood-brain barrier
Iron
Transferrin
Divalent Metal Transporter 1
Ferroportin
HFE
Restless Legs Syndrome
Neurodegenerative Disease - Abstract:
- Iron is one of the most important micronutrients due to its involvement as a cofactor in various essential processes in the body such as oxygen transport and the electron transport chain. For neuroscientists, iron is of critical interest because of its function in both neurotransmitter synthesis and myelination. The study of iron in the brain has revealed both gradual accumulation as we age and also dyshomeostasis in disease. Regulation of iron movement into the brain is managed by a blood-brain barrier (BBB). The BBB is comprised of the microvasculature throughout the brain and is characterized by tight junctions that allow for regulation of nutrient transport between the blood and the brain. Despite the importance of iron in brain function, research on the mechanism by which iron enters the brain is limited. To date, there are two potential mechanisms that have been proposed: (i) transcytosis of the iron transporter, transferrin (Tf) and (ii) endocytosis and release into the cell through divalent metal transporter 1 (DMT-1). Our research efforts have revealed both mechanisms are present. Two mechanisms may be required because the direct transcytosis mechanism overlooks the iron needs of the endothelium and the second mechanism identifies a pathway by which the endothelium and the brain iron needs are met. The overall hypothesis for this thesis is that iron movement into the brain is regulated by the brain. Both cell culture and animal models were utilized to examine the two key questions regarding brain iron uptake that were investigated: (i) what are the mechanisms of iron movement into the brain and (ii) how is iron movement into the brain regulated. In the first subset of cell culture studies, we demonstrated increased release of iron from the endothelium when exposed to apo-Tf (iron poor), however hepcidin, a protein known to block iron export by binding to the iron export protein ferroportin, had no impact on iron release alone or on iron release induced by apo-Tf. These data identify iron poor transferrin in extracellular fluid in the brain as a potential mechanism to stimulate iron release from the BBB. The second set of studies focused on the impact of both apo-Tf and DMT-1 on transport of iron from the blood side of the cultured model to the brain side. A DMT-1 inhibitor, XEN602, was implemented to investigate the role of DMT-1 in iron transport. Neither apo-Tf nor XEN602 had a significant effect on total transport of 59Fe or Tf. Of note, however, is that XEN602 exposure did not result in increases in iron within the cells, which suggests that a majority of the iron seen in the brain-side chamber was transcytosed when the endocytic model was blocked. Given, that the first set of studies demonstrated regulation of iron release and transport in the BBB model, we next sought to evaluate how a neurological disorder that was associated with brain iron deficiency would impact iron transport and release. Specifically, we obtained cerebrospinal fluid (CSF) from patients with restless legs syndrome (RLS) to test the hypothesis that the brain iron deficiency in RLS results in increased signaling to upregulate brain iron uptake. First, we found that CSF from both control and RLS patients who had low hemoglobin relative to controls had decreased transport of iron across the BBB model. The low hemoglobin RLS group was also associated with higher iron transport than the low hemoglobin non-RLS control group. Moreover, iron release had a modest but statistically significant positive correlation to systemic hemoglobin levels, but there was no correlation of transport to either hemoglobin or serum ferritin. The latter results are important because serum ferritin levels are used clinically to identify RLS patients as candidates for intravenous iron therapy. The data from the cell culture models strongly suggest, consistent with our hypothesis, that there are signals on the brain side (e.g. CSF) of the BBB that regulated iron movement across or from the BBB. Therefore, we examined 3-month old mice carrying an H67D mutation in the HFE gene. This mutation promotes excess iron accumulation in most organs of the body and in its extreme case is associated with hemochromatosis. We found that the H67D/H67D mice had elevated total brain iron levels, but took up comparable amounts of iron into the brain to the wildtype mice over 24-hour and 5-day time periods. This finding supports the hypothesis that brain iron transport is regulated by the brain even in the presence of a mutation that promotes iron uptake. We also observed significantly more 59Fe uptake into the female brain when compared to the male brain irrespective of genotype. Notably, this study is unique in that it considers the microvasculature as an independent compartment of the brain. This investigation identified iron uptake and retention within the microvessels over a 5-day time period, substantiating the hypothesis that the BBB can function as an iron reservoir for the brain. Together, the data presented in this thesis confirm that the BBB functions as a central regulatory hub for brain iron uptake. We have demonstrated that there is regulation of release of iron that comes from the brain side of the BBB. The data with the mutant mice suggest that once the brain growth has plateaued, brain iron uptake is constant even if there is a mutation present that promotes uptake. There is also a significant sex effect for brain iron uptake. The data in this thesis provide new insights into regulation of iron uptake into the brain and, because the signals for brain iron status come from the brain, the data call into question the use of iron supplementation strategies to improve cognitive impairment beyond the developmental stage.