The objective of this study was to determine the regulation of Fth1- and Tf-bound iron uptake into the brain by apo- and holo-Tf in vivo. In pursuit of this aim, we discovered significant sex differences in the regulation of iron uptake mediated by these two proteins. The results of this study have demonstrated that the ratio of apo- to holo-Tf in the CSF regulates Tf-bound iron uptake in males, but not in females in this model. However, there was significant variation in 55Fe-Tf uptake in females. To address these differences, we performed ovariectomies aimed to determine if reducing circulating estrogen would enable the regulatory response to apo- and holo-Tf infusions that were seen in males. We found that reducing peripheral estrogen did not change the lack of response of 55Fe-Tf uptake into MVs and release into the brain following infusion of apo- or holo-Tf. However, the variability that had been seen in the intact females was significantly reduced to that seen in males after removal of circulating estrogen. Additionally, delivery of Fth1 bound iron was not responsive to the ratio of apo- to holo-Tf in the CSF of either males or females. A particularly notable finding in this study was that MVs contained significantly more of the injected iron regardless of the delivery protein than the brain parenchyma even though the MVs account for only 2% of the total brain cells10. This finding further establishes our position that the ECs serve as a reservoir for iron for subsequent regulated release into the brain. Previous studies reporting on uptake of iron or other nutrients have rarely differentiated what is in the microvasculature versus what has entered the brain parenchyma. Furthermore, our data demonstrate that acquisition of brain iron is dependent on carrier protein and sex.
Previously, we and other have postulated the concept of regulation of iron release to the brain by endothelial cells of the BBB in cell culture models13–16, 24,25. For example, Simpson et al. demonstrated that CSF from iron deficient monkeys, as well as conditioned media from iron chelated astrocytes, increased iron release from bovine retinal endothelial cells16 in a bi-chamber model of the BBB. Moreover, our group previously showed, using iPSC-derived ECs in a simulated BBB model, that exposure to apo-Tf resulted in increases in both 59Fe-Tf and 59Fe-Fth1 transport from apical to basal chambers, whereas incubation with holo-Tf decreased their transport14. In vitro conditions simulating iron deficient environments have repeatedly resulted in increased iron transport across the BBB16,26,27. However, until now, the demonstration of in vivo regulation was lacking. Our in vivo data from male mice support regulated release of iron from ECs forming the MV and suggest that the brain uses apo- and holo-Tf to relay its iron status to ECs, which in turn release more or less iron in response. An example of how this feedback can occur in situ is that, following iron uptake by neurons and astrocytes, these cells release apo-Tf into the extracellular fluid28,29. Thus, areas of greater energetic activity can regionally signal for increased iron release from MVs. Thus, our data address for the first time local regulation of brain iron uptake in response to iron utilization and help explain the findings of Beard et al. who demonstrated that brain iron uptake differs in various regions30.
The role of Fth1 as an iron delivery protein to the brain is a relatively new concept with great implications as it binds nearly 2000 times more iron than Tf31. It has been reported that Fth1 can replace Tf as the iron delivery protein for oligodendrocytes32 and ECs14. Fth1 is a substantial iron contributor to the brain during development, as up to postnatal day 22, mice take up significantly more Fth1 bound iron than Tf bound iron into the brain18. In previous in vitro studies, the iron status of Tf in the basal compartment of the BBB model impacted the amount of Fth1-bound iron that was transported across the ECs14. However, in this in vivo study, we did not see any significant differences in Fth1 bound iron uptake into MVs or release into the brain following infusion of apo- or holo-Tf. In females, the infusion of apo-Tf did result in a two-fold increase in iron release into the brain compared to sham control. Although this difference did not reach statistical significance, the Cohen’s d effect size between sham control and apo-Tf is 0.65, indicating a moderate effect. The absence of statistical significance was likely due to the variability in the different groups. Thus, the data suggest that Fth1 delivered iron is responsive to CSF iron deficiency in females.
In a few experiments conducted, infusion of aCSF increased iron uptake into MVs and release into the brain. Based on our calculations, the 0.25 µl/hour infusion rate would have resulted in an approximately 1% dilution of total CSF and, thus, should have minimal effect on endogenous Tf signals with complete turnover every 1.8 hours in the mouse33. It is possible that in the less than 1 µl volume of the mouse lateral ventricle34 this initial dilution is locally greater and results in a more regional iron uptake and release. Regardless, the observation that apo-Tf or aCSF increases the uptake of transferrin-bound iron to the brain and release by the microvasculature underscores how exquisitely fined tuned the signaling from the brain to the MVs regarding iron status is.
Significant sex differences were detected in baseline (sham control group) iron uptake between Tf and Fth1. Female mice took up significantly more iron bound to Tf than to Fth1, while there was no statistically significant difference in iron uptake by either delivery protein in males. There was an increased proportion of Tf-bound iron released into the brain in females relative to males, indicating that iron was more rapidly released to the brain. The differences in baseline uptake would suggest differences in iron levels in the brain but studies have shown there is little to no difference in total brain iron levels between males and females35,36. These studies, however, largely fail to examine the process of iron accumulation. Brain iron accumulation was addressed by Duck et al., who showed that 24 hours after injecting mice with 59Fe-Tf, males and females had the same amount of iron uptake; however, after five days post injection, females took up significantly more 59Fe than males20. Combined with our data presented herein, these findings indicates that females have more iron uptake over time than males. More iron accumulation by females compared to males would be consistent with increased in myelin turnover37 and dopamine synthesis38,39 reported in females; both processes are dependent on iron as a co-factor40,41. The constant utilization of iron for these metabolic processes likely leads to an increased requirement of iron uptake into the brain which seems to be predominantly met by regulation of Fth1. This idea is also consistent with the lack of Tf delivered iron response by females to the infusion of apo- and holo-Tf. Future studies to decipher how differences in metabolic needs impact female brain iron uptake are needed.
In conclusion, this study is the first demonstration of in vivo regulation of brain iron uptake into MVs and subsequent release into the brain by apo- and holo-Tf. Moreover, we have identified striking sex differences in the baseline uptake and regulation of iron uptake for both Tf and Fth1. Understanding the sex differences and differences in Tf versus Fth1 delivered iron is crucial for clinical translation of these studies for the treatment of brain iron dysregulation and use for drug delivery efforts.