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1.
FASEB J ; 38(1): e23331, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-38031991

RESUMO

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.


Assuntos
Deficiências de Ferro , Ferro , Camundongos , Masculino , Animais , Feminino , Ferro/metabolismo , Camundongos Endogâmicos C57BL , Encéfalo/metabolismo , Transferrina , Ferro da Dieta/metabolismo
2.
J Biol Chem ; 299(2): 102868, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36603765

RESUMO

Iron is essential for normal brain development and function. Hence, understanding the mechanisms of iron efflux at the blood-brain barrier and their regulation are critical for the establishment of brain iron homeostasis. Here, we have investigated the role of exosomes in mediating the transfer of H-ferritin (FTH1)- or transferrin (Tf)-bound iron across the blood-brain barrier endothelial cells (BBBECs). Our study used ECs derived from human-induced pluripotent stem cells that are grown in bicameral chambers. When cells were exposed to 55Fe-Tf or 55Fe-FTH1, the 55Fe activity in the exosome fraction in the basal chamber was significantly higher compared to the supernatant fraction. Furthermore, we determined that the release of endogenous Tf, FTH1, and exosome number is regulated by the iron concentration of the endothelial cells. Moreover, the release of exogenously added Tf or FTH1 to the basal side via exosomes was significantly higher when ECs were iron loaded compared to when they were iron deficient. The release of exosomes containing iron bound to Tf or FTH1 was independent of hepcidin regulation, indicating this mechanism by-passes a major iron regulatory pathway. A potent inhibitor of exosome formation, GW4869, reduced exosomes released from the ECs and also decreased the Tf- and FTH1-bound iron within the exosomes. Collectively, these results indicate that iron transport across the blood-brain barrier is mediated via the exosome pathway and is modified by the iron status of the ECs, providing evidence for a novel alternate mechanism of iron transport into the brain.


Assuntos
Barreira Hematoencefálica , Exossomos , Ferro , Humanos , Barreira Hematoencefálica/metabolismo , Células Endoteliais/metabolismo , Exossomos/metabolismo , Ferro/metabolismo , Transferrina/metabolismo , Transporte Biológico
3.
FASEB J ; 37(12): e23307, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37983646

RESUMO

Glioblastoma is one of the deadliest malignancies facing modern oncology today. The ability of glioblastoma cells to diffusely spread into neighboring healthy brain makes complete surgical resection nearly impossible and contributes to the recurrent disease faced by most patients. Although research into the impact of iron on glioblastoma has addressed proliferation, there has been little investigation into how cellular iron impacts the ability of glioblastoma cells to migrate-a key question, especially in the context of the diffuse spread observed in these tumors. Herein, we show that increasing cellular iron content results in decreased migratory capacity of human glioblastoma cells. The decrease in migratory capacity was accompanied by a decrease in cellular polarization in the direction of movement. Expression of CDC42, a Rho GTPase that is essential for both cellular migration and establishment of polarity in the direction of cell movement, was reduced upon iron treatment. We then analyzed a single-cell RNA-seq dataset of human glioblastoma samples and found that cells at the tumor periphery had a gene signature that is consistent with having lower levels of cellular iron. Altogether, our results suggest that cellular iron content is impacting glioblastoma cell migratory capacity and that cells with higher iron levels exhibit reduced motility.


Assuntos
Neoplasias Encefálicas , Glioblastoma , Humanos , Glioblastoma/metabolismo , Movimento Celular/genética , Encéfalo/metabolismo , Linhagem Celular Tumoral , Neoplasias Encefálicas/metabolismo , Proliferação de Células
4.
Stroke ; 54(11): 2886-2894, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37750297

RESUMO

BACKGROUND: Intracerebral hemorrhage (ICH) is characterized by bleeding into the brain parenchyma. During an ICH, iron released from the breakdown of hemoglobin creates a cytotoxic environment in the brain through increased oxidative stress. Interestingly, the loss of iron homeostasis is associated with the pathological process of other neurological diseases. However, we have previously shown that the H63D mutation in the homeostatic iron regulatory (HFE) gene, prevalent in 28% of the White population in the United States, acts as a disease modifier by limiting oxidative stress. The following study aims to examine the effects of the murine homolog, H67D HFE, on ICH. METHODS: An autologous blood infusion model was utilized to create an ICH in the right striatum of H67D and wild-type mice. The motor recovery of each animal was assessed by rotarod. Neurodegeneration was measured using fluorojade-B and mitochondrial damage was assessed by immunofluorescent numbers of CytC+ (cytochrome C) neurons and CytC+ astrocytes. Finally, the molecular antioxidant response to ICH was quantified by measuring Nrf2 (nuclear factor-erythroid 2 related factor), GPX4 (glutathione peroxidase 4), and FTH1 (H-ferritin) levels in the ICH-affected and nonaffected hemispheres via immunoblotting. RESULTS: At 3 days post-ICH, H67D mice demonstrated enhanced performance on rotarod compared with wild-type animals despite no differences in lesion size. Additionally, H67D mice displayed higher levels of Nrf2, GPX4, and FTH1 in the ICH-affected hemisphere; however, these levels were not different in the contralateral, non-ICH-affected hemisphere. Furthermore, H67D mice showed decreased degenerated neurons, CytC+ Neurons, and CytC+ astrocytes in the perihematomal area. CONCLUSIONS: Our data suggest that the H67D mutation induces a robust antioxidant response 3 days following ICH through Nrf2, GPX4, and FTH1 activation. This activation could explain the decrease in degenerated neurons, CytC+ neurons, and CytC+ astrocytes in the perihematomal region, leading to the improved motor recovery. Based on this study, further investigation into the mechanisms of this neuroprotective response and the effects of the H63D HFE mutation in a population of patients with ICH is warranted.


Assuntos
Antioxidantes , Fator 2 Relacionado a NF-E2 , Animais , Camundongos , Hemorragia Cerebral/genética , Proteína da Hemocromatose/genética , Ferro/metabolismo , Mutação , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo
5.
J Neurochem ; 167(2): 248-261, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37667496

RESUMO

Excessive brain iron accumulation is observed early in the onset of Alzheimer's disease, notably prior to widespread proteinopathy. These findings suggest that increases in brain iron levels are due to a dysregulation of the iron transport mechanism at the blood-brain barrier. Astrocytes release signals (apo- and holo-transferrin) that communicate brain iron needs to endothelial cells in order to modulate iron transport. Here we use iPSC-derived astrocytes and endothelial cells to investigate how early-disease levels of amyloid-ß disrupt iron transport signals secreted by astrocytes to stimulate iron transport from endothelial cells. We demonstrate that conditioned media from astrocytes treated with amyloid-ß stimulates iron transport from endothelial cells and induces changes in iron transport pathway proteins. The mechanism underlying this response begins with increased iron uptake and mitochondrial activity by the astrocytes, which in turn increases levels of apo-transferrin in the amyloid-ß conditioned astrocyte media leading to increased iron transport from endothelial cells. These novel findings offer a potential explanation for the initiation of excessive iron accumulation in early stages of Alzheimer's disease. What's more, these data provide the first example of how the mechanism of iron transport regulation by apo- and holo-transferrin becomes misappropriated in disease that can lead to iron accumulation. The clinical benefit from understanding early dysregulation in brain iron transport in AD cannot be understated. If therapeutics can target this early process, they could possibly prevent the detrimental cascade that occurs with excessive iron accumulation.

6.
J Biomed Sci ; 30(1): 36, 2023 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-37277838

RESUMO

BACKGROUND: Apo- (iron free) and holo- (iron bound) transferrin (Tf) participate in precise regulation of brain iron uptake at endothelial cells of the blood-brain barrier. Apo-Tf indicates an iron-deficient environment and stimulates iron release, while holo-Tf indicates an iron sufficient environment and suppresses additional iron release. Free iron is exported through ferroportin, with hephaestin as an aid to the process. Until now, the molecular mechanisms of apo- and holo-Tf influence on iron release was largely unknown. METHODS: Here we use a variety of cell culture techniques, including co-immunoprecipitation and proximity ligation assay, in iPSC-derived endothelial cells and HEK 293 cells to investigate the mechanism by which apo- and holo-Tf influence cellular iron release. Given the established role of hepcidin in regulating cellular iron release, we further explored the relationship of hepcidin to transferrin in this model. RESULTS: We demonstrate that holo-Tf induces the internalization of ferroportin through the established ferroportin degradation pathway. Furthermore, holo-Tf directly interacts with ferroportin, whereas apo-Tf directly interacts with hephaestin. Only pathophysiological levels of hepcidin disrupt the interaction between holo-Tf and ferroportin, but similar hepcidin levels are unable to interfere with the interaction between apo-Tf and hephaestin. The disruption of the holo-Tf and ferroportin interaction by hepcidin is due to hepcidin's ability to more rapidly internalize ferroportin compared to holo-Tf. CONCLUSIONS: These novel findings provide a molecular mechanism for apo- and holo-Tf regulation of iron release from endothelial cells. They further demonstrate how hepcidin impacts these protein-protein interactions, and offer a model for how holo-Tf and hepcidin cooperate to suppress iron release. These results expand on our previous reports on mechanisms mediating regulation of brain iron uptake to provide a more thorough understanding of the regulatory mechanisms mediating cellular iron release in general.


Assuntos
Hepcidinas , Transferrina , Humanos , Transferrina/metabolismo , Hepcidinas/metabolismo , Células Endoteliais/metabolismo , Células HEK293
7.
J Neurooncol ; 164(3): 569-586, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37812288

RESUMO

PURPOSE: Iron plays a crucial role in various biological mechanisms and has been found to promote tumor growth. Recent research has shown that the H-ferritin (FTH1) protein, traditionally recognized as an essential iron storage protein, can transport iron to GBM cancer stem cells, reducing their invasion activity. Moreover, the binding of extracellular FTH1 to human GBM tissues, and brain iron delivery in general, has been found to have a sex bias. These observations raise questions, addressed in this study, about whether H-ferritin levels extrinsic to the tumor can affect tumor cell pathways and if this impact is sex-specific. METHODS: To interrogate the role of systemic H-ferritin in GBM we introduce a mouse model in which H-ferritin levels are genetically manipulated. Mice that were genetically manipulated to be heterozygous for H-ferritin (Fth1+/-) gene expression were orthotopically implanted with a mouse GBM cell line (GL261). Littermate Fth1 +/+ mice were used as controls. The animals were evaluated for survival and the tumors were subjected to RNA sequencing protocols. We analyzed the resulting data utilizing the murine Microenvironment Cell Population (mMCP) method for in silico immune deconvolution. mMCP analysis estimates the abundance of tissue infiltrating immune and stromal populations based on cell-specific gene expression signatures. RESULTS: There was a clear sex bias in survival. Female Fth1+/- mice had significantly poorer survival than control females (Fth1+/+). The Fth1 genetic status did not affect survival in males. The mMCP analysis revealed a significant reduction in T cells and CD8 + T cell infiltration in the tumors of females with Fth1+/- background as compared to the Fth1+/+. Mast and fibroblast cell infiltration was increased in females and males with Fth1+/- background, respectively, compared to Fth1+/+ mice. CONCLUSION: Genetic manipulation of Fth1 which leads to reduced systemic levels of FTH1 protein had a sexually dimorphic impact on survival. Fth1 heterozygosity significantly worsened survival in females but did not affect survival in male GBMs. Furthermore, the genetic manipulation of Fth1 significantly affected tumor infiltration of T-cells, CD8 + T cells, fibroblasts, and mast cells in a sexually dimorphic manner. These results demonstrate a role for FTH1 and presumably iron status in establishing the tumor cellular landscape that ultimately impacts survival and further reveals a sex bias that may inform the population studies showing a sex effect on the prevalence of brain tumors.


Assuntos
Apoferritinas , Glioblastoma , Humanos , Masculino , Feminino , Animais , Camundongos , Apoferritinas/genética , Apoferritinas/metabolismo , Ferritinas/genética , Ferritinas/metabolismo , Glioblastoma/genética , Microambiente Tumoral , Ferro/metabolismo
8.
J Anim Sci ; 1012023 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-36629252

RESUMO

Iron deficiency anemia is a significant problem in piglets, as they are born with insufficient iron stores for supporting their rapid body growth. Further, sows' milk contains inadequate iron levels for meeting the demands of piglet rapid growth in the pre-wean stage. The forms of iron present in the milk are essential to understanding bioavailability and potential routes for supplementing iron to mitigate iron deficiency anemia in piglets. Recently, our studies showed that H-ferritin (FTH1) is involved in iron transport to different tissues and can be used as an oral iron supplement to correct iron deficiency in rats and monkeys. In this study, we investigate the FTH1 levels in colostrum and milk in Yorkshires-crossbred sows (n = 27) and collected samples at the 1st, 15th, and 28th days of lactation to measure FTH1. Colostrum and milk were found to have FTH1, but there is no significant difference between the different days of lactation. FTH1 has been observed to be enriched in extracellular vesicles (EVs) of other species, and therefore examined the EVs in the samples. Colostrum-derived EVs were enriched with L-ferritin compared to FTH1, while in milk-derived EVs, only FTH1 was detected (P = 0.04). In milk-derived EVs, FTH1 was significantly higher (P = 0.021; P = 006) than FTH1 in colostrum-derived EVs. Furthermore, FTH1 levels of milk-derived EVs were significantly higher (P = 0.0002; P = 0004) than whole milk and colostrum FTH1. These results indicate that FTH1 is enriched in the milk-derived EVs and suggest that EVs play a predominant role in the FTH1 delivery mechanism for the piglet. The extent to which FTH1 in EVs accounts for the overall iron delivery mechanism in piglets is yet to be determined.


Colostrum and milk are the primary sources of nutrition for lactating mammals. Iron is an essential nutrient for nursing mammals. Piglets are routinely iron deficient and do not obtain adequate iron from sows' milk further contributing to anemia observed in young pigs. Additional information about the proteins that carry iron from the sow's breast milk to understand the bioavailability of iron and potential routes for reducing the incidence of anemia in offspring are clearly needed. We have discovered that H-ferritin (FTH1) is a potent iron transport protein and is not limited to iron storage as previously thought. Therefore, our objective was to determine whether the FTH1 is present in the sow's colostrum and milk. Furthermore, there are extracellular vesicles released from cells that are known to transport FTH1 and are reportedly present in sows' milk. Our study showed that FTH1 was present in the colostrum and milk and enriched in the milk-derived EVs. This study reveals a new protein and mechanism for iron delivery during lactation in sows that may be targeted to decrease iron deficiency in piglets.


Assuntos
Anemia Ferropriva , Doenças dos Suínos , Gravidez , Animais , Suínos , Feminino , Ratos , Leite , Colostro , Apoferritinas , Ferro , Anemia Ferropriva/veterinária , Suplementos Nutricionais , Lactação , Ração Animal/análise
9.
Res Sq ; 2023 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-36711476

RESUMO

Background : Apo- (iron free) and holo- (iron bound) transferrin (Tf) participate in precise regulation of brain iron uptake at endothelial cells of the blood-brain barrier. Apo-Tf indicates an iron deficient environment and stimulates iron release, while holo-Tf indicates an iron sufficient environment and suppresses additional iron release. Free iron is exported through ferroportin, with hephaestin as an aid to the process. Until now, the molecular mechanism of apo- and holo-Tf's influence on iron release was largely unknown. Methods : Here we use a variety of cell culture techniques, including co-immunoprecipitation and proximity ligation assay, in iPSC-derived endothelial cells and HEK 293 cells to investigate the mechanism of apo- and holo-Tf's influence over iron release. We placed our findings in physiological context by further deciphering how hepcidin played a role in this mechanism as well. Results : We demonstrate that holo-Tf induces the internalization of ferroportin through the established ferroportin degradation pathway. Furthermore, holo-Tf directly binds to ferroportin, whereas apo-Tf directly binds to hephaestin. Only pathological levels of hepcidin disrupt the interaction between holo-Tf and ferroportin, and no amount of hepcidin disrupts the interaction between apo-Tf and hephaestin. The disruption of the holo-Tf and ferroportin interaction by hepcidin is due to hepcidin's ability to rapidly internalize ferroportin compared to holo-Tf. Conclusions : These novel findings provide a molecular mechanism for apo- and holo-Tf regulation of iron release from endothelial cells. They further demonstrate how hepcidin impacts these protein-protein interactions, and offer a model for how holo-Tf and hepcidin corporate to suppress iron release. We have established a more thorough understanding of the mechanisms behind iron release regulation with great clinical impact for a variety of neurological conditions in which iron release is dysregulated.

10.
bioRxiv ; 2023 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-36712094

RESUMO

Background: Apo- (iron free) and holo- (iron bound) transferrin (Tf) participate in precise regulation of brain iron uptake at endothelial cells of the blood-brain barrier. Apo-Tf indicates an iron deficient environment and stimulates iron release, while holo-Tf indicates an iron sufficient environment and suppresses additional iron release. Free iron is exported through ferroportin, with hephaestin as an aid to the process. Until now, the molecular mechanism of apo- and holo-Tf's influence on iron release was largely unknown. Methods: Here we use a variety of cell culture techniques, including co-immunoprecipitation and proximity ligation assay, in iPSC-derived endothelial cells and HEK 293 cells to investigate the mechanism of apo- and holo-Tf's influence over iron release. We placed our findings in physiological context by further deciphering how hepcidin played a role in this mechanism as well. Results: We demonstrate that holo-Tf induces the internalization of ferroportin through the established ferroportin degradation pathway. Furthermore, holo-Tf directly binds to ferroportin, whereas apo-Tf directly binds to hephaestin. Only pathological levels of hepcidin disrupt the interaction between holo-Tf and ferroportin, and no amount of hepcidin disrupts the interaction between apo-Tf and hephaestin. The disruption of the holo-Tf and ferroportin interaction by hepcidin is due to hepcidin's ability to rapidly internalize ferroportin compared to holo-Tf. Conclusions: These novel findings provide a molecular mechanism for apo- and holo-Tf regulation of iron release from endothelial cells. They further demonstrate how hepcidin impacts these protein-protein interactions, and offer a model for how holo-Tf and hepcidin corporate to suppress iron release. We have established a more thorough understanding of the mechanisms behind iron release regulation with great clinical impact for a variety of neurological conditions in which iron release is dysregulated.

11.
bioRxiv ; 2023 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-37292926

RESUMO

Excessive brain iron accumulation is observed in early in the onset of Alzheimer's disease, notably prior to widespread proteinopathy. These findings suggest that increases in brain iron levels are due to a dysregulation of the iron transport mechanism at the blood-brain barrier. Astrocytes release signals (apo- and holo-transferrin) that communicate brain iron needs to endothelial cells in order to modulate iron transport. Here we use iPSC-derived astrocytes and endothelial cells to investigate how early-disease levels of amyloid-ß disrupt iron transport signals secreted by astrocytes to stimulate iron transport from endothelial cells. We demonstrate that conditioned media from astrocytes treated with amyloid-ß stimulates iron transport from endothelial cells and induces changes in iron transport pathway protein levels. The mechanism underlying this response begins with increased iron uptake and mitochondrial activity by the astrocytes which in turn increases levels of apo-transferrin in the amyloid-ß conditioned astrocyte media leading to increased iron transport from endothelial cells. These novel findings offer a potential explanation for the initiation of excessive iron accumulation in early stages of Alzheimer's disease. What's more, these data provide the first example of how the mechanism of iron transport regulation by apo- and holo-transferrin becomes misappropriated in disease to detrimental ends. The clinical benefit from understanding early dysregulation in brain iron transport in AD cannot be understated. If therapeutics can target this early process, they could possibly prevent the detrimental cascade that occurs with excessive iron accumulation. Significance Statement: Excessive brain iron accumulation is hallmark pathology of Alzheimer's disease that occurs early in the disease staging and before widespread proteinopathy deposition. This overabundance of brain iron has been implicated to contribute to disease progression, thus understandingthe mechanism of early iron accumulation has significant therapeutic potential to slow to halt disease progression. Here, we show that in response to low levels of amyloid-ß exposure, astrocytes increase their mitochondrial activity and iron uptake, resulting in iron deficient conditions. Elevated levels of apo (iron free)-transferrin stimulate iron release from endothelial cells. These data are the first to propose a mechanism for the initiation of iron accumulation and the misappropriation of iron transport signaling leading to dysfunctional brain iron homeostasis and resultant disease pathology.

12.
Fluids Barriers CNS ; 19(1): 49, 2022 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-35689283

RESUMO

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.


Assuntos
Ferro , Transferrina , Animais , Apoferritinas , Transporte Biológico , Encéfalo/metabolismo , Feminino , Ferro/metabolismo , Masculino , Camundongos , Transferrina/metabolismo
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