Aging is associated with increased brain iron through cortex-derived hepcidin expression
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Evaluation Summary:
Accumulation of redox-active iron in the brain is a significant cause of neurotoxicity in neurodegenerative diseases of old age. Thus, this manuscript could be of interest to neuroscientists, iron biologists, and those studying mechanisms of aging as it provides some new mechanistic insight on the role of age-related increases in hepcidin in brain iron accumulation. The current study demonstrates increased cytosolic and mitochondrial non-heme iron only in the aging brain, increased local hepcidin expression, and decreased levels of FPN1, together supporting a hypothesis that local brain hepcidin sequesters iron in neuronal cells and is associated with aging.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Abstract
Iron is an essential molecule for biological processes, but its accumulation can lead to oxidative stress and cellular death. Due to its oxidative effects, iron accumulation is implicated in the process of aging and neurodegenerative diseases. However, the mechanism for this increase in iron with aging, and whether this increase is localized to specific cellular compartment(s), are not known. Here, we measured the levels of iron in different tissues of aged mice, and demonstrated that while cytosolic non-heme iron is increased in the liver and muscle tissue, only the aged brain cortex exhibits an increase in both the cytosolic and mitochondrial non-heme iron. This increase in brain iron is associated with elevated levels of local hepcidin mRNA and protein in the brain. We also demonstrate that the increase in hepcidin is associated with increased ubiquitination and reduced levels of the only iron exporter, ferroportin-1 (FPN1). Overall, our studies provide a potential mechanism for iron accumulation in the brain through increased local expression of hepcidin, and subsequent iron accumulation due to decreased iron export. Additionally, our data support that aging is associated with mitochondrial and cytosolic iron accumulation only in the brain and not in other tissues.
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Evaluation Summary:
Accumulation of redox-active iron in the brain is a significant cause of neurotoxicity in neurodegenerative diseases of old age. Thus, this manuscript could be of interest to neuroscientists, iron biologists, and those studying mechanisms of aging as it provides some new mechanistic insight on the role of age-related increases in hepcidin in brain iron accumulation. The current study demonstrates increased cytosolic and mitochondrial non-heme iron only in the aging brain, increased local hepcidin expression, and decreased levels of FPN1, together supporting a hypothesis that local brain hepcidin sequesters iron in neuronal cells and is associated with aging.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested …
Evaluation Summary:
Accumulation of redox-active iron in the brain is a significant cause of neurotoxicity in neurodegenerative diseases of old age. Thus, this manuscript could be of interest to neuroscientists, iron biologists, and those studying mechanisms of aging as it provides some new mechanistic insight on the role of age-related increases in hepcidin in brain iron accumulation. The current study demonstrates increased cytosolic and mitochondrial non-heme iron only in the aging brain, increased local hepcidin expression, and decreased levels of FPN1, together supporting a hypothesis that local brain hepcidin sequesters iron in neuronal cells and is associated with aging.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Sato et al present a very well written manuscript focused on exploring the mechanism underlying brain aging, specifically related to excess iron. While increased iron in the aging brain has previously been demonstrated in both animal models and humans, until the current work, no evidence of mechanism has been found. The current work presents a compelling story of the important finding that localized hepcidin expression leads to iron sequestration by downregulating FPN1. However, the authors do not reconcile how this work can be explained in the context of Lu et al 2017 where no difference in FPN1 expression was observed. Furthermore, recent data on this subject warrants discussion. In addition, a more robust evaluation of TFR2 expression is unregulated in the aging brain and why / whether relatively …
Reviewer #1 (Public Review):
Sato et al present a very well written manuscript focused on exploring the mechanism underlying brain aging, specifically related to excess iron. While increased iron in the aging brain has previously been demonstrated in both animal models and humans, until the current work, no evidence of mechanism has been found. The current work presents a compelling story of the important finding that localized hepcidin expression leads to iron sequestration by downregulating FPN1. However, the authors do not reconcile how this work can be explained in the context of Lu et al 2017 where no difference in FPN1 expression was observed. Furthermore, recent data on this subject warrants discussion. In addition, a more robust evaluation of TFR2 expression is unregulated in the aging brain and why / whether relatively ineffective erythropoiesis (also associated with aging) may be driving iron loading in the liver, muscle, and brain. Finally, it is not clear whether hepcidin may be able to traverse the BBB at least unidirectionally. Some additional questions and clarifications are delineated below.
Please add to the discussion how to reconcile the current results with those of Lu et al 2017 mentioned in the introduction as showing no change in brain FPN1 expression.
Can the authors present data on erythropoiesis-related parameters in the young vs aged mice? Specifically, if the data is showing that ALAD and Hox1 are increased, these may be related to anemia in this model. As a consequence of an already known association between aging and anemia of unknown significance, an anemia associated with ineffective erythropoiesis, it is conceivable that ERFE is increased and leads to relatively suppressed hepcidin despite iron loading in the liver. Can the authors present data on erythropoiesis in the aged vs young mice, along with spleen size, and bone marrow ERFE expression?
Where in the brain is hepcidin produced? What specific cells express FPN1? These are important questions to reconcile the data presented with prior work in the field.
The authors have too quickly discounted a role for TFR2 in the brain. Several manuscripts exist in both mouse models (Pellegrino Sci Rep 2016) and GBM in humans (Voth J Clin Neurosci 2015) to promote a reason to explore this further. Worthy of further discussion and exploration.
Please edit the discussion to remove general knowledge about iron regulation and focus specifically on brain iron. Several manuscripts have been published recently from the group in Case Western on this subject (Chaudhary J Alz Dis 2021) that warrant discussion.
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Reviewer #2 (Public Review):
Levels of non-heme iron are shown to be elevated in both the mitochondria and the cytoplasm of old mice relative to young mice. Among iron-related genes, hepcidin expression is notably upregulated in aged brain. Hepcidin triggers ubiquitination and degradation of ferroportin, the only known cellular iron exporter. Accordingly, it is shown that ferroportin protein levels are decreased and ubiquitinated ferroportin in increased in aged brains. The study is interesting, but currently of limited impact.
Major limitations:
Analysis is done only at the whole brain level. The is no cell-level analysis, so it's unclear which cell types might be producing hepcidin, degrading their ferroportin, or accumulating iron.
No mechanistic information explaining hepcidin upregulation is provided.
Additional limitations:
Mice: …
Reviewer #2 (Public Review):
Levels of non-heme iron are shown to be elevated in both the mitochondria and the cytoplasm of old mice relative to young mice. Among iron-related genes, hepcidin expression is notably upregulated in aged brain. Hepcidin triggers ubiquitination and degradation of ferroportin, the only known cellular iron exporter. Accordingly, it is shown that ferroportin protein levels are decreased and ubiquitinated ferroportin in increased in aged brains. The study is interesting, but currently of limited impact.
Major limitations:
Analysis is done only at the whole brain level. The is no cell-level analysis, so it's unclear which cell types might be producing hepcidin, degrading their ferroportin, or accumulating iron.
No mechanistic information explaining hepcidin upregulation is provided.
Additional limitations:
Mice: it is unclear why the UM-HET3 mouse strain was chosen, and why only female mice were studied. Were the females nulliparous? What form of iron was in the diet, and how much?
It is not stated which part of the brain was dissected for the studies
There is no validation of the purity/enrichment in the mitochondrial fractionation
Aconitase activity is the only measure of oxidative stress employed. Additional markers should be tested for corroboration.
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Reviewer #3 (Public Review):
The manuscript by Sato et al. addresses an important question related to brain iron accumulation with aging. This is a timely question because iron accumulates in the brains of aged individuals and is believed to contribute to neurodegenerative diseases such as AD and PD.
Major strengths of the study are the question itself, a combination of techniques to measure brain iron, whether upregulation of local hepcidin is the main cause of iron accumulation, and if upregulation of hepcidin gene is responsible for the increase in protein expression. The authors also evaluate the expression of other iron modulating proteins, including Fpn, the downstream effector hepcidin.
Weaknesses include lack of robust data to support the author's claims. In many instances, the results do not support the conclusions drawn by the …
Reviewer #3 (Public Review):
The manuscript by Sato et al. addresses an important question related to brain iron accumulation with aging. This is a timely question because iron accumulates in the brains of aged individuals and is believed to contribute to neurodegenerative diseases such as AD and PD.
Major strengths of the study are the question itself, a combination of techniques to measure brain iron, whether upregulation of local hepcidin is the main cause of iron accumulation, and if upregulation of hepcidin gene is responsible for the increase in protein expression. The authors also evaluate the expression of other iron modulating proteins, including Fpn, the downstream effector hepcidin.
Weaknesses include lack of robust data to support the author's claims. In many instances, the results do not support the conclusions drawn by the authors.
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