Elsevier

Neurobiology of Aging

Volume 96, December 2020, Pages 33-42
Neurobiology of Aging

Regular article
Estrogen deficiency is associated with brain iron deposition via upregulation of hepcidin expression in aged female mice

https://doi.org/10.1016/j.neurobiolaging.2020.08.010Get rights and content

Highlights

  • Iron and hepcidin levels increased in the brains of normal aged females.

  • Estrogen reduced the levels of iron and hepcidin in the aged female brains.

  • Estrogen reduced hepcidin mRNA in the brain endothelial cells treated with lipopolysaccharide.

  • Estrogen reduced iron levels and free radicals in endothelial cells treated with lipopolysaccharide.

Abstract

The total iron level in the brain increases with age, and excess iron is associated with neurodegenerative diseases; however, the mechanism of brain iron deposition is unknown. In peripheral cells, the expression of hepcidin, a master regulator of iron homeostasis, is regulated by estrogen. This study aimed to determine whether hepcidin was involved in iron deposition in the brain and brain endothelial cells of estrogen-deficient aged female mice. Aged mice showed increased levels of hepcidin and ferritin in the brain and brain microvessels compared with young mice, and these levels were reduced by estrogen replacement in ovariectomized aged mice. In the brain endothelial cell line bEnd.3, the lipopolysaccharide (10 ng/mL)-induced increases of hepcidin mRNA and protein levels, the number of Prussian blue–positive cells, and free radicals were reduced after estrogen treatment. These results suggest that estrogen deficiency with an increase of hepcidin is partly responsible for iron deposition in the brain and brain endothelial cells and that hepcidin can be a target to prevent brain aging and neurodegeneration in postmenopausal women.

Introduction

Iron is essential for normal brain function, but its excess causes oxidative stress and has a role in neurodegenerative disease (Ward et al., 2014). The iron level should be finely regulated, but it progressively increases in the brain with age, and its level correlates with cognitive decline in the elderly, indicating dysregulation of iron homeostasis in the aged brain (Peters et al., 2015).

The cause of brain iron accumulation with aging is uncertain, but it has been suggested that estrogen affects serum iron status in women. In postmenopausal women, serum ferritin was more markedly increased than that in menstruating premenopausal women (Masse et al., 2004), and postmenopausal women taking oral hormone replacement therapy (HRT) showed lower serum ferritin levels than those not on HRT (Penckofer and Schwertz, 2000). Correspondingly, an inverse change was observed between serum ferritin and estrogen levels in healthy women during menopausal transition (Jian et al., 2009). These studies suggest that estrogen has a role in the regulation of iron status in women.

Hepcidin, which is mainly synthesized in the liver, is known as a master regulator of iron availability in the circulation and the tissues by binding and degradation of an iron exporter, ferroportin, in target cells (Nemeth et al., 2004b). When serum or tissue iron levels increase, hepcidin is induced and it inhibits iron efflux resulting in iron retention in the cells. In addition, chronic infection and inflammation upregulate hepcidin expression to reduce iron availability for microorganisms, and thereby, anemia of inflammation with high hepcidin levels is commonly observed in patients with chronic infection/inflammation (Nemeth et al., 2004a; Weiss et al., 2019). As normal aging accompanies chronic low-grade inflammation (inflammaging), hepcidin may cause dysregulation of iron metabolism in the elderly (Fairweather-Tait et al., 2014).

Transcription of hepcidin by iron and inflammation is mainly regulated by bone morphogenetic protein (BMP)/small mothers against decapentaplegic (Smad) pathway, which recognizes BMP responsive elements in the promoter of the hepcidin gene. Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway is also involved in hepcidin expression by binding to STAT3 responsive element of hepcidin promoter by inflammatory stimuli, such as lipopolysaccharide (LPS) or interleukin-6 (IL-6). However, the BMP/Smad pathway is required for IL-6–mediated hepcidin expression, suggesting a pivotal role of BMP/Smad for hepcidin expression under inflammation (Casanovas et al., 2009).

Recent studies have reported that in addition to inflammation, estrogen modulates hepcidin levels in human serum and immortalized cell lines. Premenopausal women were found to have a lower concentration of serum hepcidin than postmenopausal women, and this level was negatively correlated with serum estrogen and iron levels during the menstrual cycle of premenopausal women (Bajbouj et al., 2018; Galesloot et al., 2011). In addition, a functional estrogen response element (ERE) was identified in the promoter region of the hepcidin gene, and estrogen inhibited hepcidin transcription via the ERE of the hepcidin gene in liver cell lines (Hou et al., 2012). These studies imply that estrogen levels affect the iron status via regulation of hepcidin expression in females.

We previously reported an increase of proinflammatory cytokines in the brain of estrogen-deficient aged female mice (Jeong et al., 2016). Based on these studies, it can be hypothesized that hepcidin has a role to play in brain iron deposition of females during aging along with estrogen deficiency and mild inflammation. Brain iron and hepcidin levels were compared between young and aged female mice to test this hypothesis, and then the effect of estrogen on iron and hepcidin levels in aged female brains and immortalized mouse brain endothelial cells (bEnd.3) exposed to low-dose LPS, mimicking age-associated mild inflammation, was examined.

Section snippets

Animals

Female C57BL/6 mice were purchased from Orient Bio Inc (Seongnam, Republic of Korea). They were maintained in a specific pathogen-free animal facility at Ewha Womans University School of Medicine on a 12-hour light/dark cycle at 22 ± 2 °C. Standard rodent chow and tap water were provided ad libitum. Experiments were performed using young adult (10–11 weeks old) and healthy aged (20 months old) mice. For serum estradiol (E2) and hepcidin measurements, 5 young mice and 18 aged mice were used (n =

Increases in systemic and brain hepcidin and iron levels of aged female mice

First, the serum estrogen level was measured and confirmed that it was significantly reduced in the aged female mice (p = 0.04 vs. young; Fig. 1A), whereas the serum hepcidin level was significantly increased in the estrogen-deficient aged female mice (p < 0.01 vs. young; Fig. 1B), supporting the increase of serum hepcidin levels in postmenopausal women (Galesloot et al., 2011; Itkonen et al., 2012). To define brain iron accumulation with aging, the total iron level was measured, and it

Discussion

This study demonstrated that the increase of brain iron level of the aged female mice was associated with upregulated hepcidin expression, and the level of hepcidin was reduced by estrogen treatment in the brain and brain microvessels of the ovariectomized aged mice. We further determined that hepcidin was suppressed at the transcriptional level by estrogen, and thereby, iron deposition with free radical production was reduced in brain endothelial cells exposed to low dose of LPS, which induced

Disclosure statement

The authors report no conflicts of interest.

CRediT authorship contribution statement

Jin A. Shin: Conceptualization, Methodology, Data curation, Funding acquisition. Hee-Sun Kim: Validation, Formal analysis, Data curation. Jihee Lee Kang: Validation, Resources, Visualization. Eun-Mi Park: Conceptualization, Validation, Formal analysis, Writing - original draft, Supervision, Funding acquisition.

Acknowledgements

This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF), South Korea grant funded by the Ministry of Education, South Korea (NRF-2015R1C1A1A01052593) and by the Korea government (MSIT), South Korea (NRF-2019R1A2C1087035 and NRF-2020R1A5A2019210).

References (46)

  • D.G. Peters et al.

    The relationship between iron dyshomeostasis and amyloidogenesis in Alzheimer's disease: two sides of the same coin

    Neurobiol. Dis.

    (2015)
  • J.A. Shin et al.

    Conserved aquaporin 4 levels associated with reduction of brain edema are mediated by estrogen in the ischemic brain after experimental stroke

    Biochim. Biophys. Acta

    (2011)
  • J.A. Shin et al.

    Visceral adipose tissue inflammation is associated with age-related brain changes and ischemic brain damage in aged mice

    Brain Behav. Immun.

    (2015)
  • J.A. Shin et al.

    Activation of classical estrogen receptor subtypes reduces tight junction disruption of brain endothelial cells under ischemia/reperfusion injury

    Free Radic. Biol. Med.

    (2016)
  • J.A. Shin et al.

    Repression of adenosine triphosphate-binding cassette transporter ABCG2 by estrogen increases intracellular glutathione in brain endothelial cells following ischemic reperfusion injury

    Neurobiol. Aging

    (2018)
  • T.A. Tishler et al.

    Premenopausal hysterectomy is associated with increased brain ferritin iron

    Neurobiol. Aging

    (2012)
  • T.Y. Tsai et al.

    Suppression of Ca(2+) influx in endotoxin-treated mouse cerebral cortex endothelial bEND.3 cells

    Eur. J. Pharmacol.

    (2015)
  • R.J. Ward et al.

    The role of iron in brain ageing and neurodegenerative disorders

    Lancet Neurol.

    (2014)
  • G. Weiss et al.

    Anemia of inflammation

    Blood

    (2019)
  • E. Bachman et al.

    Testosterone suppresses hepcidin in men: a potential mechanism for testosterone-induced erythrocytosis

    J. Clin. Endocrinol. Metab.

    (2010)
  • E. Bachman et al.

    Testosterone induces erythrocytosis via increased erythropoietin and suppressed hepcidin: evidence for a new erythropoietin/hemoglobin set point

    J. Gerontol. A Biol. Sci. Med. Sci.

    (2014)
  • K. Bajbouj et al.

    Elevated levels of estrogen suppress hepcidin synthesis and enhance serum iron availability in premenopausal women

    Exp. Clin. Endocrinol. Diabetes

    (2018)
  • L.A. Beggs et al.

    Testosterone alters iron metabolism and stimulates red blood cell production independently of dihydrotestosterone

    Am. J. Physiol. Endocrinol. Metab.

    (2014)
  • Cited by (7)

    View all citing articles on Scopus
    View full text