Background: The brain requires iron for a number of processes, including energy production. Inadequate or excessive amounts of iron can be detrimental and lead to a number of neurological disorders. As such, regulation of brain iron uptake is required for proper functioning. Understanding both the movement of iron into the brain and how this process is regulated is crucial to both address dysfunctions with brain iron uptake in disease and successfully use the transferrin receptor uptake system for drug delivery. Methods: Using in vivo steady state infusions of apo- and holo-transferrin into the lateral ventricle, we demonstrate the regulatory effects of brain apo- and holo-transferrin ratios on the delivery of radioactive ⁵⁵Fe bound to transferrin or H-ferritin in male and female mice. In discovering sex differences in the response to apo- and holo-transferrin infusions, ovariectomies were performed on female mice to interrogate the influence of circulating estrogen on regulation of iron uptake. Results: Our model reveals that apo- and holo-transferrin significantly regulate iron uptake into the microvasculature and subsequent release into the brain parenchyma and their ability to regulate iron uptake is significantly influenced by both sex and type of iron delivery protein. Furthermore, we show that cells of the microvasculature act as reservoirs of iron and release the iron in response to cues from the interstitial fluid of the brain. Conclusions: These findings extend our previous work to demonstrate that the regulation of brain iron uptake is influenced by both the mode in which iron is delivered and sex. These findings further emphasize the role of the microvasculature in regulating brain iron uptake and the importance of cues regarding iron status in the extracellular fluid.

Adequate and timely delivery of iron is essential for brain development. The uptake of transferrin‐bound (Tf) iron into the brain peaks at the time of myelination, whereas the recently discovered H‐ferritin (FTH1) transport of iron into the brain continues to increase beyond the peak in myelination. Here, we interrogate the impact of dietary iron deficiency (ID) on the uptake of FTH1‐ and Tf‐bound iron. In the present study, we used C57BL/6J male and female mice at a developing (post‐natal day (PND) 15) and adult age (PND 85). In developing mice, ID results in increased iron delivery from both FTH1 and Tf for both males and females. The amount of iron uptake from FTH1 was higher than the Tf and this difference between the iron delivery was much greater in females. In contrast, in the adult model, ID was associated with increased brain iron uptake by both FTH1 and Tf but only in the males. There was no increased uptake from either protein in the females. Moreover, transferrin receptor expression on the microvasculature as well as whole brain iron, and H and L ferritin levels revealed the male brains became iron deficient but not the female brains. Last, under normal dietary conditions, ⁵⁵ Fe uptake was higher in the developing group from both delivery proteins than in the adult group. These results indicate that there are differences in iron acquisition between the developing and adult brain for FTH1 and Tf during nutritional ID and demonstrate a level of regulation of brain iron uptake that is age and sex‐dependent.

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

High-grade brain tumors such as grade III astrocytoma and glioblastoma represent among the most difficult to manage malignancies facing oncological practice. Diffuse migration and invasion into adjacent healthy brain tissue yields complete surgical resection of these tumors unfeasible. As a result, despite receiving maximal surgical resection and an aggressive course of chemoradiation, the majority of high-grade brain tumor patients suffer from recurrent disease. Increased expression levels of the homeostatic iron regulator gene (HFE) in high-grade brain tumors have been correlated with poorer outcomes. HFE is known to influence cellular iron metabolism by inhibiting transferrin-mediated iron uptake yet little is known regarding how HFE or iron impact the migratory capabilities of high-grade brain tumor cells. In order to better understand how HFE expression and cellular iron metabolism influence cell migration in high grade brain tumors, we utilized brain tumor cell lines that had been genetically manipulated to express different levels of HFE. We observed that knocking down HFE in KR158 or LN229 glioma cell lines resulted in significantly decreased migratory capacity. Since HFE is known to inhibit transferrin mediated iron uptake, we studied how directly modulating the iron status of glioma cells impacted their ability to migrate. Treatment of a panel of glioma cell lines: LN229, T98G, U87, KR158, with iron in the form of hemin or ferric ammonium citrate resulted in significantly reduced migration. Furthermore, the iron-induced reduction in migration could be rescued by the addition of deferoxamine, an iron chelator. Cell viability in response to the iron treatments was assayed and found to not be significantly altered - suggesting that cellular iron status was influencing migratory capacity independent of cell viability. To gain mechanistic insights into HFE and iron-induced effects on cell migration, we analyzed the Chinese Glioma Genome Atlas (CGGA) for correlations between HFE and the Rho GTPases RHOA, RAC1, and CDC42 – genes which are known to play a crucial role in determining the migratory capacity of cancer cells. Interestingly, we found statistically significant correlations between the Rho GTPases RHOA, RAC1, CDC42 and HFE in both grade III astrocytoma and glioblastoma patient cohorts. Immunoblotting of iron treated glioma cell lines demonstrated that expression of RhoA and Cdc42 was reduced suggesting that alterations in Rho GTPase expression and signaling may play a role in iron-induced effects on cell migration. Our results demonstrate that targeting cancer cell iron metabolism as an addition to existing treatment regimens may be a promising avenue for further investigation. Citation Format: Ganesh Shenoy, Katie Troike, Kondaiah Palsa, Madison Kuhn, Quinn Wade, Becky Slagle-Webb, Amanda Snyder, Chachrit Khunsriraksakul, Justin D. Lathia, Dhimant Desai, Hong-Gang Wang, Elizabeth Proctor, James R. Connor. The role of cellular iron and the homeostatic iron regulator (HFE) in high-grade brain tumor cell migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2430.