Role of ezrin–radixin–moesin proteins in the regulation of inflammatory cytokine expression in activated microglia

Abstract

The ERM protein family, comprising ezrin, radixin, and moesin, mediates membrane-cytoskeleton interactions, regulating key cellular functions. This study investigated the expression of ERM proteins in microglia surrounding amyloid plaques in App^NL-F knock-in mice and their role in inflammatory responses. Moesin was predominantly localized to plaque-associated microglia, and in vitro knockdown experiments revealed distinct roles of ERM proteins in cytokine regulation. These findings suggest that each ERM protein contributes to microglial function through distinct mechanisms. Further elucidating the roles of individual ERM proteins in microglial function may lead to the identification of novel therapeutic targets for neurodegenerative diseases.

Keywords

Alzheimer's disease amyloid-β ERM protein microglia neuroinflammatory

Introduction

The ezrin–radixin–moesin (ERM) proteins constitute a widely distributed family of membrane-associated proteins that link plasma membrane proteins to filamentous actin (F-actin) in the cell cortex. This linkage is important for regulating cell shape, protein localization, membrane transport, and signal transduction.¹ Among them, moesin is broadly expressed, particularly in monocytes/macrophages, neurons, and tumor cells, and is the dominant ERM protein in microglia, the brain's resident immune cells.^2,3

Microglia are activated in response to CNS damage or pathogens, producing pro-inflammatory cytokines and neurotoxic factors like nitric oxide.^4–7 This can lead to chronic neuroinflammation, contributing to neurodegenerative diseases such as Alzheimer's disease (AD).^5,6 In AD, amyloid-β (Aβ) plaques accumulate and trigger inflammatory responses.⁸ Therefore, targeting neuroinflammation by modulating these mediators may offer a promising therapeutic approach for neurodegenerative diseases.

Proteomic and histological studies have shown that Aβ plaques in early-onset AD and Down syndrome are enriched in ezrin and moesin, with moesin highly abundant in plaque-associated microglia.⁹ This finding has also been confirmed in 5xFAD mouse brains and in human AD.^9,10 Moesin also plays a role in lipopolysaccharide (LPS)-induced immune responses. Blocking moesin reduces TNFα release in LPS-stimulated immune cells, although its knockdown in microglia has shown opposite effects.^10–12 This study confirmed that ERM proteins in microglia are involved in LPS-induced inflammatory responses and investigated the distinct roles of ezrin, radixin, and moesin.

Methods

Animals

All animal experiments were conducted by the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the protocols were approved by the Committee for Animal Research at Gifu Pharmaceutical University. App^{NL−F/NL−F} (App^NL-F) knock-in (KI) mice were obtained from Dr Takashi Saito and Dr Takaomi C Saido (RIKEN BRC).¹³ In this study, we used male App^NL-F KI mice in all experiments of this study, and these animals were acclimated to and maintained at 23°C in a 12-h light/dark cycle. Mice were placed in standard laboratory cages and had free access to food and water.

Sample preparation and immunofluorescence staining

Brain sections were prepared according to a previous study.^14,15 Further information can be found in the Supplemental Methods. Male mice aged 15 months were used. Brain sections were incubated with rabbit antibodies (Abs) to moesin (1:300, Cell Signaling Technology, 3150) and with goat antibodies to Iba-1 (1:200, Abcam, ab5076), mouse antibodies S100β (1:250, Sigma-Aldrich, S2532) or mouse antibodies GFAP (1:300, Sigma-Aldrich, MAB360). For amyloid plaque staining, 1-Fluoro-2,5-bis(3-carboxy-4-hydroxystyryl)benzene (FSB) was used at a concentration of 0.0005% (Dojindo). The primary Abs were detected using Alexa Fluor 546 donkey anti-rabbit, Alexa Fluor 488 goat anti-mouse, Alexa Fluor 647 donkey anti-goat (1:500, Thermo Fisher Scientific, A10040, A11029, A10040).

Confocal images were acquired using a confocal laser scanning microscope (LSM 900, Carl Zeiss). Fluorescence intensity was quantified using the ImageJ software. Graphs and statistical analyses were generated using R software. Further information can be found in the Supplemental Methods.

Cell culture and treatment

BV-2 mouse microglial cells were purchased from Banca Biologica e Cell Factory (Genova, Italy) and maintained in Dulbecco's modified Eagle's medium (DMEM) (Fujifilm Wako) supplemented with 10% fetal bovine serum (FBS) under a humidified atmosphere containing 5% CO₂ at 37°C.

For drug treatment, BV-2 cells were seeded on 6-well plates or 24-well plates. Twenty-four hours after seeding, the culture medium was replaced with serum-free DMEM containing 0, 0.01, 0.1, and 1 µg/mL LPS (Sigma-Aldrich).

Small interfering RNA (siRNA) transfection

For the Ezrin (Ezr), Radixin (Rdx), and Moesin (Mzn) knockdown experiments, BV-2 cells were transfected with siRNA. Further information can be found in the Supplemental Methods. The siRNAs were as follows: ON-TARGETplus Non-targeting Control siRNAs (siNC, Cat# D-001810-01), ON-TARGETplus EZR SMARTpool siRNA (siEzr, Cat# L-046568-01), ON-TARGETplus RDX SMARTpool siRNA (siRdx, Cat# L-047230-01), or ON-TARGETplus MSN SMARTpool siRNA (siMsn, Cat# L-044428-01).

RNA preparation and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

RNA preparation and RT-qPCR were performed as described previously.¹⁶ Further information can be found in the Supplemental Methods. The primers used in the RT-qPCR analysis were as follows: Gapdh (forward: 5′-CCTCGTCCCGTAGACAAAATG -3′; reverse: 5′-TCTCCACTTTGCCACTGCAA -3′), Ezr (forward: 5′-AGAAGCGGATCACAGAGGCA -3′; reverse: 5′-CGCAGCGTCTTATACTTGTCCC-3′), Rdx (forward: 5′-GTGATGAACCACCGAAGCGA-3′; reverse: 5′-GGCCGGCCTTGACATTCTCA -3′), Msn (forward: 5′-CCTGGAAAATGAGAAGAAGAAGCGT -3′; reverse: 5′-TCAAGTTCTAGGGCCCTGCG -3′), Tnfα (forward: 5′- GTTCTATGGCCCAGACCCTCA-3′; reverse: 5′- GCCTTGTCCCTTGAAGAGAACC-3′), and Il-1β (forward: 5′- TCCACGATGAGGACATGAGCAC-3′; reverse: 5′- GAACGTCACACACCAGCAGGTTA-3′).

Western blotting

Western blot was performed as described previously.¹⁷ Further information can be found in the Supplemental Methods. The primary antibodies used in the immunoblotting were as follows: anti-ezrin (1:1000, Cell Signaling Technology, #3145), anti-radixin (1:1000, Sigma-Aldrich, R3653), anti-moesin (1:1000, Cell Signaling Technology, #3150), or anti-β-actin (1:5000, Sigma-Aldrich, A1978).

Statistical analysis

Data are presented as means ± standard error of the mean (SEM). Statistical comparisons were made using R studio. Further information can be found in the Supplemental Methods.

Results

Previous studies have shown that in App^NL-F KI mice, cortical Aβ deposition begins by 6 months of age, and cognitive functions are impaired by the age of 18 months.¹⁸ In this study, we prepared brain sections from the cerebral cortex of 15-month-old App^NL-F KI mice and analyzed the relationship between amyloid plaques and ERM proteins using fluorescent immunohistochemistry (Figure 1). Among the ERM proteins, moesin exhibited strong staining around FSB-positive amyloid deposition (Figure 1(A)–(D), (a)–(d)). Moesin was also observed in cells with a morphology resembling activated ameboid microglia. Fluorescent immunohistochemistry using moesin and Iba-1, a marker of microglia, revealed that moesin co-localized with Iba-1-positive microglia (Figure 1(a)–(d)). Moesin was colocalized not only with Iba-1-positive microglia around FSB-positive amyloid deposits (FSB+), but also with Iba-1-positive microglia in FSB-negative regions (FSB−) (Figure 1(e)–(h)). Iba-1-positive microglia in FSB-negative regions exhibited a morphology resembling ramified microglia. Co-localization of moesin with S100β-positive astrocytes was also observed, but it was not as strong as the co-localization with Iba-1-positive microglia (Figure 1(E)–(H)). To evaluate differences in moesin expression between microglia surrounding FSB-positive amyloid deposits and those in FSB-negative regions, fluorescence intensities were compared. The results demonstrated that moesin was more strongly expressed in microglia associated with FSB-positive deposits (Figure 1(M)). Also, we prepared brain sections from the cerebral cortex of 15-month-old wild-type (WT) mice. Iba-1-positive ramified microglia were observed throughout the cortex, and the microglia were found to co-localize with a weak signal of moesin (Figure 1(I)–(L), (i)–(l)). To evaluate differences in moesin expression between microglia in FSB-negative regions of App^NL-F KI mice and microglia in WT mice, fluorescence intensities were compared. As a result, no significant difference was observed between the two groups (Figure 1(N)). Ezrin and radixin did not show around FSB-positive amyloid deposition (data not shown).

Figure 1.

Moesin is expressed by microglia in the brains of 15-month-old App^NL-F KI and WT mice. (A–D) Representative immunofluorescence images of moesin (A), Iba-1 (B), and FSB (C) in the cortex from 15-month-old App^NL-F KI mice. (a–h) Enlarged immunofluorescence images of A–D. (E–H) Representative immunofluorescence images of moesin (E), S100β (F), and FSB (G) in the cortex from 15-month-old App^NL-F KI mice. (I–L) Representative immunofluorescence images of moesin (I), Iba-1 (J), and FSB (K) in the cortex from 15-month-old wild-type mice. (i–l) Enlarged immunofluorescence images of I–L. (M) In App^NL-F KI mouse samples (n = 3), quantification of moesin expression was performed in microglia located around FSB-positive amyloid deposits and in microglia in FSB-negative regions. (N) In WT mouse samples (n = 3) and App^NL-F KI mouse samples (n = 3), quantification of moesin expression was performed in microglia from WT mice and in microglia located in FSB-negative regions of App^NL-F KI mice. Scale bars: 50 μm (A–L), 10 μm (a–l). Data are presented as the means ± standard error of the mean (SEM). Statistical analysis was performed using a paired t-test for panel (M) and Welch's t-test for panel (N) (n = 3 animals, *p < 0.05).

Based on our findings that ERM proteins, particularly moesin, are present in microglia adjacent to Aβ deposits in the App^NL-F KI mouse brain, we conducted in vitro functional studies using BV-2 cells, a mouse cell line of microglia, to investigate the effects of ERM protein knockdown on microglial inflammatory cytokine expression. Microglia are known to increase the expression of pro-inflammatory cytokines in response to stimulation by Aβ.⁸ BV-2 cells were used as an in vitro model due to their ability to mimic key microglial functions such as cytokine production and phagocytosis. First, we examined the expression of ERM proteins in BV-2 cells. The results demonstrated that BV-2 cells expressed all three ERM proteins—ezrin, moesin, and radixin—consistent with our previous findings (Figure 2(A)–(D)).³ Furthermore, LPS treatment, a known activator of microglia, significantly increased ezrin mRNA and protein levels, whereas moesin showed a significant mRNA increase but no significant protein change, although an increasing trend was observed (Figure 2(A), (C), (D); Supplemental Figure 2A, C). In contrast, no changes were observed in either mRNA or protein levels of radixin upon LPS treatment (Figure 2(B), (D); Supplemental Figure 2B).

Figure 2.

Changes in ERM proteins in BV-2 cells following treatment with LPS. (A-D) Ezrin, radixin, moesin, and β-actin protein levels were determined at 24 h after LPS treatment. Data are presented as mean ± S.E.M. Statistical significance was determined using the one-way ANOVA, followed by the Tukey test (n = 3, **p < 0.01, ***p < 0.001 versus 0 µg/mL LPS).

Next, to assess the genetic changes resulting from ERM protein knockdown in BV-2 cells, we evaluated the gene expression levels of ezrin, radixin, and moesin by RT-qPCR following siRNA-mediated knockdown of each gene. In BV-2 cells transfected with siEzr, ezrin expression was reduced by approximately 70% (Figure 3(A)). Similarly, in BV-2 cells transfected with siRdx and siMsn, radixin and moesin expression levels were reduced by approximately 70% and 80%, respectively (Figure 3(B), (C)). In addition, western blot analysis confirmed that these proteins were also knocked down at the protein level (Figure 3(D)–(G)). These results confirm that ezrin, radixin, and moesin were effectively knocked down by their respective siRNA treatments. Furthermore, no compensatory upregulation among the ERM proteins was observed in the knockdown cells (Figure 3(A)–(G)).

Figure 3.

ERM protein knockdown impacts inflammatory cytokine production by BV-2 cells. In-vitro knockdown experiments using BV-2 cells treated with siMsn, siRdx, siMoe, or siNC. (A–C) Results from RT-qPCR experiments demonstrating the efficiency of knockdown by each siRNA as compared to siNC. (D–G) Results from western blot experiments demonstrating the efficiency of knockdown at the protein level for each siRNA as compared to siNC. (H–I) Bar graphs showing cytokine data obtained by RT-qPCR 24 h after LPS treatment following transfection with siEzr, siRdx, siMsn, or siNC: (H) TNFα, (I) IL-1β. Data are presented as mean ± S.E.M. Statistical significance was determined using the one-way ANOVA, followed by the Tukey test (n = 4, *p < 0.05, **p < 0.01, ***p < 0.001 versus siNC). non-targeting control; NC.

BV-2 cells exhibited a significant increase in the expression of TNFα and IL-1β following LPS treatment (Supplemental Figure 3). Furthermore, in BV-2 cells transfected with siMsn, TNFα expression, but not IL-1β expression was significantly upregulated in response to LPS stimulation compared to cells transfected with siNC (Figure 3(H) and (I)). In contrast, TNFα and IL-1β expression levels were significantly reduced in BV-2 cells transfected with siRdx compared to those transfected with siNC (Figure 3(H) and (I)). Notably, transfected with siEzr had no effect on LPS-induced cytokine expression in BV-2 cells (Figure 3(H) and (I)).

Discussion

In this study, it was confirmed that moesin was more strongly expressed in activated ameboid microglia surrounding amyloid deposits in App^NL-F KI mice (Figure 1). Furthermore, in vitro experiments, BV-2 cells express the ERM proteins ezrin, radixin, and moesin, and it is thought that these may play an essential role in cytokine expression in microglia activated by LPS (Figures 2 and 3). Specifically, in BV-2 cells, LPS stimulation increased ezrin and moesin mRNA, but not radixin. At the protein level, only ezrin increased significantly (Figure 2; Supplemental Figure 2). In BV-2 cells in which moesin was knocked down, LPS stimulation caused an increase in the expression of TNFα, while in cells in which radixin was knocked down, the expression of both TNFα and IL-1β decreased. In BV-2 cells in which ezrin was knocked down, there was no change in cytokine expression (Figure 3). Although the detailed mechanism remains unclear, it is thought that ERM proteins play an essential role in the regulatory mechanism of cytokine expression, at least for cytokines such as TNFα and IL-1β, in activated microglia.

Aβ plaques in early-onset AD and DS are enriched in ezrin and moesin, with moesin being highly abundant in plaque-associated microglia in both 5xFAD mice and AD.^9,10 In our in vivo experiments, we observed strong signals for moesin around amyloid plaques in App^NL-F KI mice, but we were unable to detect any signals for ezrin. We also found that moesin is expressed in Iba-1-positive microglia. In vitro experiments, LPS treatment of BV-2 cells significantly increased ezrin at both mRNA and protein levels, whereas moesin increased only at the mRNA level. The lack of a significant increase in moesin at the protein level may be due to post-transcriptional regulation, such as reduced translational efficiency, enhanced protein degradation, or altered subcellular localization limiting detection under our extraction conditions.

In this experiment, no compensatory changes in ERM protein expression were observed in each knockdown cell. These results suggest that ERM proteins may play different roles in microglia. In this study, we investigated the role of ERM proteins by focusing on inflammatory cytokine expression. As in the previous paper,¹⁰ the knockdown of moesin increased LPS-induced TNFα expression. These results suggest that moesin may have an inhibitory effect on inflammatory cytokine expression in activated microglia. On the other hand, in BV-2 cells in which radixin was knocked down, cytokine expression in response to LPS was significantly suppressed. This result suggests that radixin may be necessary for basic cytokine expression. Ezrin increased in response to LPS but did not affect cytokine expression. This result suggests that ezrin may be involved in functions of microglia other than cytokine expression.

In conclusion, this study suggests that each ERM proteins contribute to the activation of microglia and the inflammatory response surrounding amyloid plaques through distinct mechanisms. However, this study is limited to LPS-induced microglia, and further detailed investigations using Aβ-induced microglia are necessary to discuss the relationship between ERM proteins and AD. Further studies are needed to explore the precise mechanisms through which ERM proteins contribute to microglial function and their potential as targets for therapeutic intervention.

Footnotes

Acknowledgements

We thank Prof. Takaomi C Saido (RIKEN BRC) and Prof. Takashi Saito (Institute of Brain Science, Nagoya City Univ.) for the App^NL-F KI mice. The breeders of App^NL-F KI mice used in this study were provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan.

ORCID iD

Masatoshi Inden

Ethical considerations

All animals were cared for in strict accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996), and the experimental design was approved by the Ethics Committee of Animal Research at Gifu Pharmaceutical University (Approval no. 2023-005) on March 15, 2023.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Author contribution(s)

Ayane Nakano: Conceptualization; Data curation; Formal analysis; Investigation; Writing – original draft; Writing – review & editing.

Shogo Maekawa: Data curation; Investigation; Writing – original draft; Writing – review & editing.

Takanori Murakami: Data curation; Investigation.

Ayaka Fujimaki: Data curation; Investigation.

Shinnosuke Takizawa: Data curation; Investigation.

Junya Murata: Data curation; Investigation.

Kazuki Ohuchi: Data curation; Investigation.

Hisaka Kurita: Data curation; Investigation.

Kazuyuki Takata: Conceptualization; Funding acquisition; Supervision; Writing – original draft; Writing – review & editing.

Masatoshi Inden: Conceptualization; Funding acquisition; Project administration; Supervision; Writing – original draft.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by a grant from the Smoking Research Foundation (Grant No. 2021G021 for MI), Takeda Science Foundation (MI) and the Kyoto Pharmaceutical University Fund for the Collaborative Research (KT).

Declaration of conflicting interests

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

Data availability statement

The data supporting the findings of this study are available within the article and its supplemental material.

Supplemental material

Supplemental material for this article is available online.

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Supplementary Material

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