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Abstract

Objective

Iron is the most abundant metal in the human brain, and plays a crucial role in many biological processes. However, disruptions in brain iron metabolism can lead to iron buildup, which occurs with aging and is linked to several brain disorders, including Alzheimer's disease. Microglia, the brain's resident immune cells, have the highest capacity to store iron, which is stored intracellularly within ferritin complexes. Importantly, women are at a higher risk of developing Alzheimer's disease and experience faster disease progression compared to men.

Methods

We used postmortem brain samples from patients with Alzheimer's disease and small vessel disease patients of both sexes for immunohistochemical studies. Samples were stained with the Prussian blue method to visualize iron deposits and with antibodies against the microglia marker Iba1 and ferritin light chain.

Results

Our study reveals that the number of iron deposits and the levels of ferritin light chain in microglia are positively correlated in men with Alzheimer's disease, but negatively correlated in women. There is no correlation between brain iron deposition and ferritin in samples from patients with small vessel disease of both sexes.

Conclusions

These results could inform more tailored approaches to the treatment and management of Alzheimer's disease based on sex-specific differences in brain iron metabolism and microglial iron storage capacity.

Keywords

Brain iron accumulation microglia ferritin sex differences

Introduction

The high-energy needs of the brain are supported by adequate levels of iron.¹ Iron, the most abundant metal in the brain,² is essential for various functions, including the electron transport chain, oxygen storage, cell proliferation and differentiation, myelogenesis, and dopamine production.³ Disruptions in brain iron metabolism are linked to several neurodegenerative disorders, such as Alzheimer's disease (AD), Huntington's disease, Parkinson's disease, and neurodegeneration with brain iron accumulation.^4–6 Increasing evidence suggests that brain iron accumulation contributes to AD's pathogenesis rather than merely a consequence of the disease, as iron accumulation is associated with amyloid plaque formation.⁷ Magnetic resonance imaging studies have shown that brain iron content increases with age and is elevated in patients with AD,^8,9 particularly in the basal ganglia, and correlates with cognitive decline.¹⁰ Higher values of quantitative susceptibility mapping (a type of magnetic resonance imaging sensitive to iron levels) in patients with AD are linked to faster disease progression and cognitive decline.¹¹ Despite these findings, the molecular mechanisms driving increased iron accumulation in the brain during aging and AD remain unclear.

Women show higher frailty than men, as women exhibit worse health conditions; however, women are less vulnerable to death than men, which is known as the “sex-frailty paradox.”¹² Women are at a higher risk of developing AD and experience faster disease progression than men when adjusted for age.^13,14 Two-thirds of all patients with AD are women,¹⁵ and women have a two-fold higher lifetime risk of developing AD compared to men,¹⁶ after adjusting for longevity.¹⁷ Women with AD exhibit enhanced cognitive decline, worse cognitive function, and accelerated brain atrophy rates compared to men.^18–20 The association between clinical manifestations of AD and AD pathology is more significant in women,²⁰ and aged women with one APOE4 allele have a four-fold increased risk of developing AD compared to male APOE4 heterozygotes.²¹ Despite these findings, the factors contributing to these sex differences in AD pathology remain incompletely understood.

The ferritin complex acts as a cellular storage of iron and the levels of ferritin are importantly regulated by intracellular iron.²² In conditions with enhanced iron in the cell, ferritin is upregulated to store iron safely. Ferritin is composed of monomers of the ferritin heavy chain and the ferritin light chain. The ferritin light chain is especially important as it confers stability to this complex.²³ Microglia have the highest iron storage capacity in the brain, and ferritin, the protein that stores iron in cells, is highly expressed in microglia, compared with other brain cells.²⁴

We hypothesize that iron deposition in the brain is dependent on the capacity of microglia to store iron and that this relationship differs between women and men with AD. In this study, we used postmortem brain samples from Asian women and men with AD. Samples were either stained with Prussian blue to visualize iron deposits or with antibodies against the microglia marker Iba1 and the ferritin component ferritin light chain. In addition, given that vascular disease contributes to Alzheimer's pathology,²⁵ we also used postmortem brain samples from small vessel disease (SVD) patients to determine if the relationship between iron deposits and ferritin in microglia between sexes is consistent between these two types of dementia. We found that the levels of ferritin in microglia and the number of iron deposits show a positive linear regression in the basal ganglia and putamen of men with AD; however, brain iron deposition negatively correlates with microglial ferritin in the brain samples from women with AD. Interestingly, the brain samples from patients with SVD do not show any correlation between microglial ferritin and brain iron deposition of either sex. This finding is important for understanding sex differences in the pathways regulating ferritin levels and iron storage capacity in microglia in the brains of women and men with AD.

Material and methods

Human samples

Brain samples (globus pallidus, putamen, neocortex, and white matter) from AD and SVD patients of both sexes (five individuals per sex and disease) were obtained from Niigata University, following the recommendations from the Declaration of Helsinki 1975. The range of sample collection is between May 1996 and August 2020. The age ranges for each group are 78 to 95 years in men with AD (mean = 85.2 ± 17); 68 to 104 in women with AD (mean = 85.2 ± 36); 70 to 92 in men with SVD (mean = 81.2 ± 22); and 76 to 90 in women with SVD (mean = 81.8 ± 14). No significant difference was found between the ages of the different groups (p = 0.84). The Braak stages in men and women with AD were between IV and VI. The cause of death in all patients with AD was pneumonia. In contrast, the cause of death in patients with SVD was more variable (pneumonia, gastrointestinal hemorrhage, cardiovascular complications, or thymic epithelial tumor). Neurological reports are available in Supplemental Table 1. The present study was approved by the Ethics Committee of Niigata University (G2018-0013: 24 October 2018, and 2022-0022: 30 May 2022). Written informed consent for autopsy including the use of tissues for research purposes was obtained from the patient’s family.

Prussian blue staining

Prussian blue staining visualizes iron in tissues in the form of ferric iron bound to ferritin.²⁶ Paraffin blocks were sliced at 5 μm thickness on a microtome. After deparaffinization, brain slices were incubated with 20% hydrochloric acid:10% potassium ferrocyanide (1:1) solution for 20 m. After washing in distilled water, sections were counterstained with nuclear fast red for 5 m. Then, slices were rinsed in distilled water and dehydrated with 95% and 100% ethanol and xylene.

We used the EVOS^TM FL Auto 2 imaging system (Thermo Fisher Scientific) to take 10 20x magnification images of the internal capsule of the globus pallidus, the putamen, the neocortex, and the white matter underneath the neocortex by an investigator blinded to groups. We used the Fiji-ImageJ software to identify the particles positive to the Prussian blue staining. For each image, the color channels were separated using the vector FastRed FastBlue DAB in the “colour deconvolution” command. Then, the same threshold was applied for the blue channel of all images to identify the Prussian blue-positive particles. Thus, the iron deposits were highlighted, and the number of iron deposits per area (mm²) and the area of each Prussian blue-positive particle were analyzed.

Immunofluorescence

After deparaffinization, brain slices (5 μm thickness) were incubated with citrate buffer solution (C9999, Millipore-Sigma, 1:10) during two cycles of 10 m at 100°C for antigen retrieval. Then, samples were washed and incubated with 0.2% bovine serum albumin/phosphate-buffered saline (PBS) as a blocking buffer solution for 1 h at room temperature. Then, slides were co-immunostained with antibodies against Iba1 (NB1028, Novus biological, 1:400) and against the ferritin light chain (ab201975, Abcam, 1:5000). Both antibodies were added into the same antibody solution and incubated overnight at 4°C. After washing samples with PBS twice, brain samples were incubated with fluorescent-tagged secondary antibodies (anti-goat-AlexaFluor^®488, ab150129, Abcam, 1:200; anti-mouse-AlexaFluor^®647, ab150115, Abcam, 1:200) for 2 h at room temperature. The autofluorescence eliminator Everbrite Trueblack Hardset Mounting medium (23018, Biotium) was applied to the samples and then mounted with Fluoroshield™ with 4',6-diamidino-2-phenylindole (F6057, Millipore-Sigma).

We used a Leica DM8i SPE confocal microscope to take 10 20x magnification images of the internal capsule of the globus pallidus and the putamen by an investigator blinded to groups. The fluorescence intensity of ferritin was analyzed with ImageJ software. Channels were split and the Iba1-positive areas in the red channel were used as regions of interest to measure the fluorescence intensity of ferritin in the green channel. The arbitrary units from the fluorescence intensity were used to calculate the relative fluorescence intensity considering the average of the fluorescence intensity in the group with male individuals as 1.

Statistical analysis

All analyses were performed using GraphPad Prism software (v.10). We used the two-tailed Mann–Whitney U test to compare means of: the number of iron deposits per mm² between men and women with AD or SVD; the percentage of the area of iron deposits between men and women with AD or SVD; the fluorescence intensity of ferritin light chain between men and women with AD or SVD; and the number of Iba1-positive particles per mm² between men and women with AD or SVD. We used the Kruskal–Wallis multiple comparisons test to compare the means of: the number of iron deposits per mm² between four brain regions (globus pallidus, putamen, neocortex, and white matter). We used simple linear regression to determine a linear relationship between the number of iron deposits/mm² and the relative fluorescence intensity of the ferritin light chain. A value of p < 0.05 was considered significantly different. Bar graphs represent the mean ± standard error of the mean (SEM).

Results

The putamen and globus pallidus, which are parts of the basal ganglia, are the brain regions with the highest accumulation of iron.²⁷ However, iron deposition in the cortex has been identified as a marker of cognitive impairment in AD.^28,29 Thus, we determined the number of iron deposits by Prussian blue staining in the basal ganglia (globus pallidus and putamen) and cortex (neocortex and white matter areas) in postmortem human samples from patients with AD or SVD. Prussian blue staining is commonly used to visualize microhemorrhages and iron deposits in tissues. We observed that the globus pallidus and putamen contained a significantly higher number of iron deposits per area, compared with the neocortex and white matter in samples from patients with AD and SVD (Figure 1(a) to (c)).

Figure 1.

Iron deposition in the globus pallidus, putamen, neocortex, and white matter of patients with AD or SVD by sex. (a) Representative images of postmortem human brain samples from patients with AD or SVD in globus pallidus, putamen, neocortex, and white matter stained with Prussian blue. Scale bar, 50 µm. (b) Quantification of the number of iron deposits per mm² in postmortem human brain samples from patients with AD of both sexes in globus pallidus, putamen, neocortex, and white matter stained with Prussian blue. (c) Quantification of the number of iron deposits per mm² in postmortem human brain samples from patients with SVD of both sexes in globus pallidus, putamen, neocortex, and white matter stained with Prussian blue. All data are mean ± SEM with n = 10 per brain region. Kruskal–Wallis test, Dunn's multiple comparisons test. (d) Representative images of postmortem brain samples from men and women with AD in the globus pallidus stained with Prussian blue. Scale bar, 100 µm. (e) Quantification of the number of iron deposits per mm² (left) and the percentage of the area positive to iron deposits (right) in the globus pallidus of men and women with AD. (f) Quantification of the number of iron deposits per mm² (left) and the percentage of the area positive to iron deposits (right) in the putamen of men and women with AD. (g) Representative images of postmortem brain samples from men and women with SVD in the globus pallidus stained with Prussian blue. Scale bar, 100 µm. (h) Quantification of the number of iron deposits per mm² (left) and the percentage of the area positive to iron deposits (right) in the globus pallidus of men and women with SVD. (i) Quantification of the number of iron deposits per mm² and the percentage of the area positive to iron deposits in the putamen of men and women with SVD. All data are mean ± SEM with n = 5 per brain region and sex. Two-tailed Mann–Whitney U test.

Then, we aimed to determine whether sex differences exist in iron deposition in the globus pallidus and putamen in samples from patients with AD or SVD. We observed an increase of iron deposits in the globus pallidus (p = 0.22) and putamen (p = 0.31) from women with AD, compared with those from men with AD; however, these differences were not significant (Figure 1(d) to (f)). In contrast, we did not find any increase in iron deposition in the globus pallidus (p = 0.69) and putamen (p = 0.42) from women with SVD, compared with those from men with the same pathology (Figure 1(g) to (i)).

Then, we determined if the levels of ferritin in microglia differ between men and women with AD or SVD. For this, postmortem brain samples from the globus pallidus and putamen of men and women with AD or SVD were stained with antibodies against Iba1 (a marker of microglia) and antibodies against the ferritin light chain. We did not use samples from the neocortex and white matter as these areas contain minimal amounts of iron deposits. We did not find differences in the number of Iba1-positive cells between sexes in the globus pallidus (p = 0.84) and putamen (p = 0.84) in samples from AD patients (Figure 2(a) to (c)). The number of Iba1-positive cells was increased, but not significantly, in the globus pallidus (p = 0.55) and putamen (p = 0.22) of women with SVD, compared with those from men with SVD (Figure 2(d) to (f)). Interestingly, the levels of ferritin in Iba1-positive cells were slightly reduced in the globus pallidus (p = 0.69) and putamen (p = 0.55) of women with AD (Figure 2(b) and (c)), and the globus pallidus (p = 0.15) and putamen (p = 0.15) of SVD (Figure 2(e) and (f)), compared with those from men with the same pathology.

Figure 2.

Correlation between microglial ferritin and brain iron deposition in patients with AD or SVD in a sex-dependent manner. (a) Representative images of postmortem brain samples from men and women with AD in the globus pallidus stained with antibodies against Iba1 (red) and ferritin light chain (green). Scale bar, 25 µm. (b) Quantification of the number of Iba1-positive cells per mm² (left) and the relative fluorescence intensity of ferritin light chain in Iba1-positive cells (right) in the globus pallidus of men and women with AD. (c) Quantification of the number of Iba1-positive cells per mm² (left) and the relative fluorescence intensity of ferritin light chain in Iba1-positive cells (right) in the putamen of men and women with AD. (d) Representative images of postmortem brain samples from men and women with SVD in the globus pallidus stained with antibodies against Iba1 (red) and ferritin light chain (green). Scale bar, 25 µm. (e) Quantification of the number of Iba1-positive cells per mm² (left) and the relative fluorescence intensity of ferritin light chain in Iba1-positive cells (right) in the globus pallidus of men and women with SVD. (f) Quantification of the number of Iba1-positive cells per mm² (left) and the relative fluorescence intensity of ferritin light chain in Iba1-positive cells (right) in the putamen of men and women with SVD. All data are mean ± SEM with n = 5 per brain region and sex. Two-tailed Mann–Whitney U test. (g) Simple linear regression between the relative fluorescence intensity of ferritin light chain in Iba1-positive cells and iron deposits per mm² in the globus pallidus of men (top) and women (bottom) with AD. (h) Simple linear regression between the relative fluorescence intensity of ferritin light chain in Iba1-positive cells and iron deposits per mm² in the putamen of men (top) and women (bottom) with AD. (i) Simple linear regression between the relative fluorescence intensity of ferritin light chain in Iba1-positive cells and iron deposits per mm² in the globus pallidus of men (top) and women (bottom) with SVD. (j) Simple linear regression between the relative fluorescence intensity of ferritin light chain in Iba1-positive cells and iron deposits per mm² in the putamen of men (top) and women (bottom) with SVD. R-squared and p-values are represented in each graph.

Given that we observed differences in the number of iron deposits and ferritin levels in Iba1-positive cells between men and women with AD or with SVD, we hypothesized that these two variables (iron deposits and microglial ferritin levels) are related in a sex-dependent manner. Thus, we used the values of iron deposits/mm² and the ferritin levels in Iba1-positive cells for a representation of linear regression in the globus pallidus and putamen for each sex and pathology. We observed a positive linear regression between iron deposits and ferritin levels in microglia in the globus pallidus (p = 0.22) and putamen (p = 0.02) in men with AD (Figure 2(g) and (h), top panel); however, women with AD showed a significant negative linear regression between these two variables in the globus pallidus (p = 0.008) and putamen (p = 0.01) (Figure 2(g) and (h), bottom panel). Interestingly, there is no significant linear regression between iron deposits and ferritin levels in the globus pallidus (p = 0.49) and putamen (p = 0.29) in men with SVD (Figure 2(i) and (j), top panel), neither in the globus pallidus (p = 0.33) and putamen (p = 0.07) in men with SVD (Figure 2(i) and (j), bottom panel). To confirm that the correlation between microglia ferritin and iron deposition is not due to the number of microglia itself, we analyze the linear regression between the particles positive to Iba1 and the number of iron deposits in the globus pallidus and putamen of men and women with AD or SVD. We did not observe a significant correlation between the number of microglia and iron deposits in either men and women with AD or SVD in the globus pallidus and putamen (Supplemental Figure 1).

Discussion

In our study, we found that the number of iron deposits and the levels of ferritin light chain in microglia showed a positive correlation in the putamen in men with AD; however, in women with AD, brain iron deposition negatively correlates with microglial ferritin in globus pallidus and putamen. Interestingly, women and men with SVD do not show any relationship between iron deposits and ferritin in microglia. This highlights that this sex difference is specific in AD and not in SVD. Furthermore, there is no significant correlation between the number of particles positive to Iba1 and the number of iron deposits in the globus pallidus and putamen of men and women with AD or SVD, suggesting that the differences in microglia ferritin and iron deposits between men and women with AD are not influenced by the number of microglia.

Magnetic resonance imaging allows researchers to identify the brain regions with higher iron content in healthy patients and patients with neurodegeneration, being globus pallidus and putamen the areas with higher susceptibility values.^30–32 However, to determine in what cell types iron accumulates, it is necessary to use molecular techniques that cannot be applied in living patients. These techniques have identified that all brain cell types have the ability to store iron in the ferritin complexes. However, aberrant accumulation of iron in astrocytes and neurons leads to cell death.³³ Importantly, microglial cells have the highest capacity to store iron. This ability allows microglia to phagocytize iron-containing debris from neurodegeneration in AD, but eventually, microglia become activated and may trigger neuroinflammation,^34,35 which negatively affects many cellular processes.

Differences in the iron storage capacity and iron metabolism between sexes have been reported in other studies with patients with AD. O’Neil et al.³⁶ found that microglia cells in women with AD have higher iron levels than microglia cells in men with AD. Interestingly, Gue et al.³⁷ found in a single-cell RNA sequencing study that the expression of the FTL gene, which codes for the ferritin light chain, is reduced in microglia cells in women with AD, compared with women without dementia, but no difference is observed between men with AD and no-dementia. Elevated levels of iron in microglia reduce its phagocytic capacity, alter microglial mitochondrial respiration, and promote the synthesis and release of pro-inflammatory cytokines, which negatively affect surrounding cells and tissues.^38–41 Pro-inflammatory microglia are activated in AD, and numerous microglia are found in brain areas associated with degenerative features.⁴⁰ Our data suggest that the reduced levels of ferritin light chain in microglia lead to enhanced iron deposition in the basal ganglia of women with AD. Altogether suggests that the reduced expression of FTL in microglia in women with AD can negatively affect the iron storage capacity of their brains, and contribute to exacerbating neuroinflammation, accelerating the AD pathology.

Our study has the limitation of the low number of postmortem brain samples from patients with AD and SVD. Importantly, our study enhances the relevance of determining whether there exist sex differences in the molecular mechanisms that regulate iron metabolism in the brain in patients with AD and how these differences contribute to the different brain iron deposition and pathology progression. The study of sex differences in iron accumulation in other brain cell types in AD will also help to define the different pathology observed between men and women with AD.

Conclusion

Our study is the first to propose that there exist sex differences in the correlation between brain iron deposition and microglial ferritin. Importantly, these sex differences occur particularly in AD pathology, but not in SVD. These findings are relevant for further research in the field of sex differences in brain iron metabolism and AD.

Supplemental Material

sj-docx-1-sci-10.1177_00368504251336080 - Supplemental material for Sex differences in brain iron deposition and microglial ferritin in Alzheimer's disease

Supplemental material, sj-docx-1-sci-10.1177_00368504251336080 for Sex differences in brain iron deposition and microglial ferritin in Alzheimer's disease by Syed Mushfiqur Rahman, Chunfeng Tan, Akiyoshi Kakita and Jose Felix Moruno-Manchon in Science Progress

Supplemental Material

sj-docx-2-sci-10.1177_00368504251336080 - Supplemental material for Sex differences in brain iron deposition and microglial ferritin in Alzheimer's disease

Supplemental material, sj-docx-2-sci-10.1177_00368504251336080 for Sex differences in brain iron deposition and microglial ferritin in Alzheimer's disease by Syed Mushfiqur Rahman, Chunfeng Tan, Akiyoshi Kakita and Jose Felix Moruno-Manchon in Science Progress

Supplemental Material

sj-jpeg-3-sci-10.1177_00368504251336080 - Supplemental material for Sex differences in brain iron deposition and microglial ferritin in Alzheimer's disease

Supplemental material, sj-jpeg-3-sci-10.1177_00368504251336080 for Sex differences in brain iron deposition and microglial ferritin in Alzheimer's disease by Syed Mushfiqur Rahman, Chunfeng Tan, Akiyoshi Kakita and Jose Felix Moruno-Manchon in Science Progress

Footnotes

Acknowledgments

We thank Lori Capozzi, Michael Maniskas, Hilda Ahnstedt, Catalina Herrera, Ashley Fordyce, and Azusa Yamada for administrative support.

ORCID iDs

Syed Mushfiqur Rahman

Chunfeng Tan

Akiyoshi Kakita

Jose Felix Moruno-Manchon

Ethical considerations

The present study was approved by the Ethics Committee of Niigata University (G2018-0013: 24 October 2018, and 2022-0022: 30 May 2022). Written informed consent for autopsy including the use of tissues for research purposes was obtained from the patient’s family.

Author contributions/CRediT

S.M.R. performed immunostaining, imaged slides, and analyzed images. C.T. advised on the study. A.K. provided samples and advised on the study. J.F.M.M. designed and wrote the manuscript. All authors have read and approved the current version of the manuscript.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was conducted with financial support from the NIA (No. R21AG075750, to J.F.M.M.), the Texas Alzheimer's Research and Care Consortium (No. 957578, to J.F.M.M.), the American Heart Association (No. 856061, No. J.F.M.M.), the Global Collaborative Research Project FY2022 from Niigata University (No. 3-NUBRI-58, to J.F.M.M.), and start-up funds from the University of Texas Health Science Center at Houston McGovern Medical School (to J.F.M.M.).

Conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data availability

Data generated from this study is available in Harvard Dataverse ().

Supplemental material

Supplemental material for this article is available online.

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Supplementary Material

Please find the following supplemental material available below.

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