Spliceosomal GTPase Eftud2 regulates microglial activation and polarization

Abstract

Elongation factor Tu GTP binding domain protein 2 (Eftud2) is a spliceosomal GTPase that serves as an innate immune modulator restricting virus infection. Microglia are the resident innate immune cells and the key players of immune response in the central nervous system. However, the role of Eftud2 in microglia has not been reported. In this study, we performed immunofluorescent staining and western blot assay and found that Eftud2 was upregulated in microglia of a 5xFAD transgenic mouse model of Alzheimer’s disease. Next, we generated an inducible microglia-specific Eftud2 conditional knockout mouse line (CX3CR1-CreER; Eftud2^f/f cKO) via Cre/loxP recombination and found that Eftud2 deficiency resulted in abnormal proliferation and promoted anti-inflammatory phenotype activation of microglia. Furthermore, we knocked down Eftud2 in BV2 microglia with siRNA specifically targeting Eftud2 and found that Eftud2-mediated regulation of microglial proinflammatory/anti-inflammatory phenotype activation in response to inflammation might be dependent on the NF-κB signaling pathway. Our findings suggest that Eftud2 plays a key role in regulating microglial polarization and homeostasis possibly through the NF-κB signaling pathway.

Introduction

Microglia serve as the most common brain-resident immune cells and are usually highly branched in the healthy brain (Nayak et al., 2014). Microglia not only participate in the regulation of neurogenesis (Arnò et al., 2014; Squarzoni et al., 2015), but also play an important role in the regulation of synaptic pruning and synaptic plasticity during brain development (Paolicelli et al., 2011; Colonna and Butovsky, 2017; Li et al., 2020). Moreover, microglia are considered to be a key regulator in the pathological processes of neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s disease, and amyotrophic lateral sclerosis, and of some psychiatric disorders (Colonna and Butovsky, 2017). Although recent studies have revealed the unique transcriptomic characteristics of microglia (Zhang et al., 2014; Hammond et al., 2019), the regulatory process and mechanisms of their post-transcriptional regulation remain unclear.

There is increasing evidence that microglial activation in the central nervous system (CNS) is heterogeneous (Cherry et al., 2014; Tang and Le, 2016). They can differentiate into the proinflammatory M1 phenotype under lipopolysaccharide (LPS) or interferon-γ stimulation, or into the anti-inflammatory M2 phenotype upon interleukin (IL)-4/IL-13 stimulation (Orihuela et al., 2016). Although it is currently believed that the balance between M1 and M2 microglia polarization plays an important role in the progression of CNS diseases, such as spinal cord injury (Kigerl et al., 2009; Cherry et al., 2014), stroke (Cherry et al., 2014), and AD, the detailed mechanisms underlying this pathological process remain unclear. Of note, researchers recently pointed out that the concept of microglial M1/M2 polarization should be discarded until the ontogeny and functional significance of microglia are elucidated (Ransohoff, 2016; Lee et al., 2021). Given these controversies, in this study, microglial activation was divided into two subtypes: an inducible nitric oxide synthase (iNOS)-positive proinflammatory phenotype and an arginase-1 (Arg1)-positive anti-inflammatory phenotype.

Alternative splicing (AS) is involved in the activation of immune cells and the regulation of immune activity (Frankiw et al., 2019; Su and Huang, 2021). The process of AS is mediated by spliceosomes composed of U1, U2, U4, U5, and U6 small nuclear ribonucleic proteins (snRNPs), as well as a series of auxiliary proteins (Wan et al., 2020). Eftud2 (also called Snu114), a key component of the spliceosome complex U5 snRNP, is a spliceosomal GTPase, which mediates spliceosome activation and reunion (Turner et al., 2004; Brenner and Guthrie, 2005; Small et al., 2006). Eftud2 was originally identified as a pathogenic gene of microcephaly with mandibulofacial dysostosis (Lines et al., 2012; Huang et al., 2016; Thomas et al., 2020). In a zebrafish model, mutations in Eftud2 led to p53-dependent apoptosis of neural progenitor cells, suggesting that Eftud2 is related to cell fate determination (Lei et al., 2017). Furthermore, Eftud2 serving as an innate immune regulator regulates the innate immune response to viral infection by regulating AS of retinoic acid inducible gene-I (Zhu et al., 2015). A recent study has also reported that specific Eftud2 knockout in myeloid can significantly inhibit the occurrence of chronic intestinal inflammation and tumor, which also indicates the critical role of Eftud2 in immune responses (Lv et al., 2019). However, the function of Eftud2 or other spliceosome components in microglia has not been reported.

Here, we initially demonstrated that Eftud2 was dramatically upregulated in microglia of both aging brain and LPS-induced inflammatory responses. Then, we generated mice with Eftud2-deficient microglia to investigate the role of Eftud2 in microglia. We found that in the absence of Eftud2, microglia exhibited increased proliferation with amoeba-like activation morphology. Furthermore, our in vitro BV2 microglia culture and in vivo genetic ablation results demonstrated that Eftud2 plays a vital role in the regulation of microglial proinflammatory and anti-inflammatory phenotype activation. Mechanistically, Eftud2 might be involved in regulating inflammatory responses through the nuclear transcription factor-κB (NF-κB) signaling pathway.

Methods

Animals

All animal experiments were approved by the Institutional Animal Care and Use Committee of Beijing Institute of Basic Medical Sciences (approval No. SYXK (Jing) 2019-0004) in September 2020. All experiments were designed and reported according to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines (Kilkenny et al., 2010). The construction of CX3CR1-CreER mice has been described previously (Yona et al., 2013). Eftud2^f/f mice were provided by Institute of Pharmacology and Toxicology (Beijing, China) (Lei et al., 2017). 5xFAD transgenic mice were provided by Beijing Institute of Basic Medical Sciences (Beijing, China) (Cheng et al., 2021). CX3CR1-CreER; Eftud2^f/f mice were obtained by crossing Eftud2^f/f mice with CX3CR1-CreER mice. Conditional knockout (cKO) mice were produced in the second generation, and Eftud2^f/f littermates were used as controls (Additional Figure 1A ^{(1.1MB, tif)} ). By crossing CX3CR1-CreER mice with Ai9 mice (Cat# 007909; JAX Laboratory, Bar Harbor, ME, USA), we generated CX3CR1-CreER; Ai9 reporter mice. CX3CR1-CreER recombinant enzyme was activated by intraperitoneal injection of 4-OH-tamoxifen (4-OH-TAM; Cat# H7904; Sigma-Aldrich, St. Louis, MO, USA). The resulting offspring were genotyped by polymerase chain reaction (PCR) using genomic DNA. The primer sequences used are listed in Table 1.

Table 1.

Primers used for genotyping

Gene name	Primer sequence (5’–3’)	Product size (bp)
Eftud2	Forward: GCA GGA AAG GTT AGC AGT C	Wild-type: 300
	Reverse: GTT CTC GTC GGT GGA ATA	Mutant: 400
CX3CR1-CreER	Forward: AGC CGG AAG CCC AAG AGC ATC	Wild-type: 200
	Forward: TGC TGC TGC CCG ACA ACC AC	Mutant: 700
	Reverse: CCG CCA GAC GCC CAG ACT A
Ai9	Forward: AAG GGA GCT G CA GTG GAG TA	Wild-type: 297
	Forward: GGC ATT AAA G CA GCG TAT CC	Mutant: 196
	Reverse: CCG AAA ATC T GT GGG AAG TC
	Reverse: CTG TTC CTG T AC GGC ATG G

Eftud2: Elongation factor Tu GTP binding domain protein 2.

Drugs and treatment

4-OH-TAM was dissolved in corn oil at 20 mg/mL and stored at 4°C. CX3CR1-CreER; Eftud2^f/f mice were injected intraperitoneally with 4-OH-TAM at a dose of 100 mg/kg body weight per day for 5 days. Briefly, the mice were injected with 4-OH-TAM at postnatal day 14 (P14) and the brain tissue was harvested at P21. Alternatively, the mice were injected with 4-OH-TAM at P30 and the brain tissue was harvested at P42 (Additional Figure 1B ^{(1.1MB, tif)} and C ^{(1.1MB, tif)} ).

Cell culture and transfection

BV2 cell line (RRID: CVCL_XD67) was obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in minimum essential media (Invitrogen, Waltham, MA, USA) containing 10% fetal bovine serum (Gibco, Grand Island, CA, USA) and 1% penicillin-streptomycin (Invitrogen). All cells were maintained at 37°C in a 5% CO₂ environment. LPS (1 μg/μL; Sigma-Aldrich; dissolved in normal saline) was used to stimulate BV2 cells in vitro for 12 or 24 hours after transfection. BV2 cells were identified through immunostaining using anti-ionized calcium binding adapter molecule 1 (Iba1). The Eftud2 and negative control short interfering RNAs (siRNAs) were synthesized by GenePharma (Suzhou, China). Lipofectamine RNAimax (Invitrogen) was used to transfect the Eftud2 or negative control siRNAs into the BV2 cells according to the manufacturer’s instructions. Briefly, siRNA and transfection reagent were mixed at a ratio of 2:1 and were added to BV2 cells after standing for 20 minutes. To ensure stable knockdown of Eftud2 in BV2 cells, supernatants or cells were collected 3 days after siRNA transfection.

Immunofluorescence staining

Immunofluorescence staining was performed to investigate the localization and expression of Eftud2, Ki67, ionized calcium-binding adaptor molecule 1 (Iba-1), Arg1, iNOS, glial fibrillary acidic protein (GFAP), and NeuN. Mice were anesthetized by intraperitoneal administration of 1% pentobarbital (50 mg/kg, China National Pharmaceutical Group Corporation, Beijing, China) and perfused with physiological saline followed by 4% paraformaldehyde. Then, the entire brain was removed and postfixed in 4% paraformaldehyde for over 24 hours. Dehydration was performed with 15% and then 30% sucrose solutions. Brain coronal slices (30 μm thick) were prepared by using a freezing microtome (Thermo Fisher Scientific, Waltham, MA, USA). For immunofluorescent staining, the sections were blocked with phosphate-buffered saline containing 3% bovine serum albumin (Solarbio, Beijing, China) and 0.3% Triton X-100 for 1 hour. The following antibodies were used overnight at 4°C: rabbit anti-Eftud2 (1:200, Abcam, Cambridge, UK, Cat# ab72456, RRID: AB_1268731), mouse anti-Ki67 (1:400, Becton Dickinson and Company, Franklin Lake, NJ, USA, Cat# 550609, RRID: AB_393778), goat anti-Iba-1 (1:400, Abcam, Cat# ab5076, RRID: AB_2224402), rabbit anti-Arg1 (1:400, Proteintech, Chicago, IL, USA, Cat# 16001-1-AP, RRID:AB_2289842), rabbit anti-iNOS (1:200, Cell Signaling Technology, Boston, MA, USA, Cat# 13120, RRID: AB_2687529), mouse anti-GFAP (1:400, Sigma-Aldrich, Cat# G3893, RRID: AB_477010), and mouse anti-NeuN (1:400, Abcam, Cat# ab104224, RRID: AB_10711040). The following secondary antibodies were added: Alexa Fluor 488-conjugated donkey anti-goat IgG (1:500, Biotium, Fremont, CA, USA, Cat# 20016, RRID: AB_10563028), Alexa Fluor 568-conjugated donkey anti-rabbit IgG (1:500, Biotium, Cat# 20098, RRID: AB_10557118), Alexa Fluor 488-conjugated donkey anti-rabbit IgG (1:500, Biotium, Cat# 20015, RRID: AB_10559669), Alexa Fluor 488-conjugated donkey anti-mouse IgG (1:500, Biotium, Cat# 20014, RRID: AB_10561327), and Alexa Fluor 568-conjugated donkey anti-mouse IgG (1:500, Biotium, Cat# 20105, RRID: AB_10557030) for 1 hour at 25°C. β-amyloid (Aβ) was labeled by Thioflavin S staining. Briefly, the Thioflavin S stain was diluted at 1:1000 in 50% absolute ethanol, and sections were incubated for 10 minutes. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (ZSGB-BIO, Beijing, China, Cat# ZLI-9556). Frozen sections of CX3CR1-CreER; Ai9 reporter mice were directly mounted with 4',6-diamidino-2-phenylindole after washing with phosphate-buffered saline. The td-Tomato⁺ cells have red fluorescence. All images were captured on an Olympus FV-1200 confocal microscope (Olympus, Center Valley, PA, USA) and analyzed with Imaris 9.3.1 software (Abingdon, Oxfordshire, UK).

Phagocytosis assay

To evaluate the phagocytic function of cells, phagocytosis assay was performed. BV2 cells were collected into 6-well plates overnight. Cells were incubated in the presence or absence of siRNA and LPS for the indicated periods. Fluorescent microspheres (Sigma-Aldrich, Cat# L3030) were then added to the treated cells for 30 minutes as a marker for liquid phase phagocytosis. Then, cells were washed three times with phosphate-buffered saline and fixed in 4% paraformaldehyde for 30 minutes. Next, cells were washed with phosphate-buffered saline and nuclei were stained with DAPI. All images were captured on a confocal microscope and analyzed with Imaris software. Phagocytic efficiency was determined as previously reported (Koenigsknecht and Landreth, 2004) using the following equation: phagocytic efficiency (%) = (1 × X₁ + 2 × X₂ + 3 × X₃…. + n × X_n)/total number of cells × 100, where X_n represents the number of cells containing n microspheres.

Western blot analysis

Western blot was performed to detect protein changes in proinflammatory and anti-inflammatory phenotype activation and the NF-κB pathway. Total protein was extracted from BV2 microglia or 5xFAD mouse brain tissues using radioimmunoprecipitation assay lysis buffer (Sigma-Aldrich, Cat# 89900). Protein concentration was measured using a bicinchoninic acid protein assay kit (Sigma-Aldrich, Cat# 23227). The proteins were isolated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes (YangguangBio, Beijing, China), which were then sealed in 5% milk and incubated overnight with primary antibodies at 25°C. The primary antibodies used for western blotting were as follows: rabbit anti-Eftud2 (1:1000, Abcam, Cat# ab72456, RRID: AB_1268731), rabbit anti-myeloid differentiation primary response gene 88 (1:1000, Cell Signaling Technology, Cat# 4283S, RRID: AB_10547882), rabbit anti-p-65 (1:1000, Cell Signaling Technology, Cat# 8242, RRID: AB_10859369), rabbit anti-phosphorylated p65 (p-p65; 1:1000, Cell Signaling Technology, Cat# 3033), rabbit anti-TLR4 (1:1000, Cell Signaling Technology, Cat# 38519S), and mouse anti-β-actin (1:1000, Sungene Biotech, Beijing, China, Cat# KM9001). The membranes were then incubated for 1 hour at 25°C with horseradish peroxidase-conjugated goat anti-mouse IgG (1:5000, ZSGB-BIO, Cat# ZB-2305, RRID: AB_2747415) or horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000, ZSGB-BIO, Cat# ZB-2301, RRID: AB_2747412). Enhanced chemiluminescence was used to detect signals on X-ray film. The signals were quantified using ImageJ 2.0.0 software (National Institutes of Health, Bethesda, MD, USA) (Schneider et al., 2012) after scanning.

Real-time polymerase chain reaction

To explore the related factors in proinflammatory and anti-inflammatory phenotype activation and the NF-κB pathway at the transcriptional level, real-time polymerase chain reaction (RT-PCR) was performed. Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen, Cat# 15596018). Next, RNA was reversed to complementary DNA and amplified by the kit. RT-PCR was conducted in triplicate using SYBR Green PCR master mix (CWBIO, Beijing, China, Cat# CW2601) with the appropriate forward and reverse primers. The instrument settings were as follows: initial denaturation for 10 minutes at 95°C and 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds. For quantitative analysis of gene expression, results were averaged from three replicates in three independent experiments. Values were normalized to β-actin levels. The primer sequences used are listed in Table 2.

Table 2.

Primers used for real-time PCR

Gene name	Primer sequence (5’–3’)
Eftud2	Forward: GAT CGA GCA TAC CTA CAC TGG C
	Reverse: GTA CAT CTT CGT CGT GTG GCA
Arg1	Forward: CTC CAA GCC AAA GTC CTT AGA G
	Reverse: AGG AGC TGT CAT TAG GGA CATC
iNOS	Forward: GTT CTC AGC CCA ACA ATA CAA GA
	Reverse: GTG GAC GGG TCG ATG TCA C
IL-10	Forward: GCT CTT ACT GAC TGG CAT GAG
	Reverse: CGC AGC TCT AGG AGC ATG TG
TNFα	Forward: CCA AGA GGT GAG TGC TTC CC
	Reverse: CTG TTG TTC AGA CTC TCT CCC T
TLR4	Forward: ATG GCA TGG CTT ACA CCA CC
	Reverse: GAG GCC AAT TTT GTC TCC ACA
MyD88	Forward: TCA TGT TCT CCA TAC CCT TGG T
	Reverse: AAA CTG CGA GTG GGG TCA G
p65	Forward: AGG CTT CTG GGC CTT ATG TG
	Reverse: TGC TTC TCT CGC CAG GAA TAC
Actb1	Forward: GGC TGT ATT CCC CTC CAT CG
	Reverse: CCA GTT GGT AAC AAT GCC ATG T

Actb1: Beta-actin; Arg1: arginase-1; Eftud2: elongation factor Tu GTP binding domain protein 2; IL-10: interleukin-10; iNOS: inducible nitric oxide synthase; MyD88: myeloid differentiation primary response gene 88; TLR4: Toll-like receptor 4; TNF-α: tumor necrosis factor-α.

Three-dimensional reconstruction of microglia

Frozen sections of Eftud2^f/f and CX3CR1-CreER; Eftud2^f/f mice brain tissue were stained with the indicated primary antibody (Iba-1; 1:400, Abcam, Cat# ab5076) at 4°C overnight. AlexaFluor 488-conjugated donkey anti-goat IgG (1:500, Biotium, Cat# 20016) was incubated at room temperature for 2 hours. Nuclei were counterstained with DAPI. Confocal images were captured using an Olympus microscope and a 60× oil immersion objective. Z-stack images with 0.5-μm intervals were taken at a depth of 30 μm. Images were further analyzed using Imaris software. Microglia branch length, number of branches, number of ends, and cell body area were analyzed.

Statistical analysis

No statistical methods were used to predetermine sample sizes; however, our sample sizes were similar to those reported in previous publications (Dudiki et al., 2020; Wang et al., 2021). No animals or data points were excluded from the analysis. All data were statistically analyzed using GraphPad Prism 8.0.2 (GraphPad, San Diego, CA, USA, www.graphpad.com). ImageJ was used for statistical analysis of fluorescence intensity and positive cell number per unit area of immunofluorescence images. For in vivo experiments, at least three mice were used for each genotype. The investigator was blind to the group allocation during the experiment and data collection. The data were presented as mean ± standard error (SEM). Statistical differences between different groups were analyzed using the two-tailed unpaired Student’s t-test. The data were considered to be significant at P < 0.05.

Results

Upregulated expression of Eftud2 in microglia-mediated inflammatory response

Previous studies have shown that Eftud2 plays an important role in macrophage inflammation (De Arras et al., 2014; Lv et al., 2019). To further determine the potential role of Eftud2 in the inflammatory responses of microglia in the brain, we first examined the expression of Eftud2 in a 5xFAD transgenic mouse model of AD. Immunoblotting indicated that Eftud2 expression in 5xFAD mice was significantly higher than that in control mice (P < 0.05; Figure 1A and B). Given that microglia are significantly activated during AD progression, we next performed immunofluorescence staining on frozen brain sections of wild-type and 5xFAD mice to determine whether the elevated expression of Eftud2 in AD mice was related to microglia (Figure 1C). Immunofluorescence staining showed that the number of Iba-1⁺Eftud2⁺ cells in 5xFAD mice was significantly higher than that in control mice (P < 0.0001; Figure 1D). Thus, elevated Eftud2 expression in 5xFAD mice might be due to the activation of microglial cells.

Figure 1.

Upregulated expression of Eftud2 in microglia-mediated inflammatory response.

(A) Representative immunofluorescence staining images of Eftud2 and β-actin in brain lysates from WT and 5xFAD mice. (B) Quantitative analysis of Eftud2 protein levels normalized to β-actin. Eftud2 expression was significantly elevated in 5xFAD mice compared with WT mice. (C) Aβ (magenta, thioflavin-S), Iba-1 (green, Alexa Fluor 488) and Eftud2 (red, Alexa Fluor 568) immunostaining in cortex of WT and 5xFAD mice. Iba-1⁺Eftud2⁺ cells were significantly increased in 5xFAD mice compared with WT mice. Scale bars: 30 μm (left), 5 μm (right). (D) Quantification of Iba-1 and Eftud2 double-positive microglia normalized to WT mice. (E) Schematic diagram of LPS-stimulated BV2 cells. (F) Representative immunofluorescence staining images of Eftud2 and β-actin in response to LPS in BV2 cells. (G) Quantitative analysis of Eftud2 protein levels normalized to β-actin. Eftud2 expression in microglia was increased, especially 24 hours after LPS stimulation, compared with unstimulated control cells. (H) RT-PCR analysis of Eftud2 mRNA levels normalized to control after exposure to LPS. The mRNA level of Eftud2 was also increased after LPS stimulation. (I) Iba-1 (green, Alexa Fluor 488), Eftud2 (red, Alexa Fluor 568) immunostaining of BV2 cells after exposure to LPS for 24 hours. Eftud2 expression was significantly increased in the nuclei after LPS treatment. Scale bars: 20 μm. (J) Immunofluorescence intensity for Eftud2 was measured. Data are presented as mean ± SEM. At least three mice were used for each genotype. For cells, at least three independent experiments were repeated. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (two-tailed unpaired Student’s t-test). DAPI: 4',6-Diamidino-2-phenylindole; Ctrl: control; Eftud2: elongation factor Tu GTP binding domain protein 2; Iba-1: ionized calcium-binding adaptor molecule 1; LPS: lipopolysaccharide; MW: molecular weight; RT-PCR: real-time polymerase chain reaction; WT: wild-type.

To investigate the role of Eftud2 in microglial activation, LPS was used to stimulate the cultured BV2 microglia at different time points (Figure 1E). Western blot analysis showed that Eftud2 expression in microglia was elevated, especially 24 hours after LPS stimulation, compared with that in unstimulated control cells (12 hours, P < 0.05; 24 hours, P < 0.001; Figure 1F and G). Consistent with these findings, RT-PCR analysis showed that Eftud2 mRNA level was also elevated after LPS stimulation (12 hours, P < 0.05; 24 hours, P < 0.001; Figure 1H). We further confirmed the expression of Eftud2 in microglia by immunofluorescence staining, which also showed significantly elevated immunopositivity of Eftud2 in the nuclei after LPS treatment compared with controls (P < 0.01; Figure 1I and J). Taken together, these results suggest that Eftud2 might be involved in the regulation of microglial activation and inflammatory response in aging and degenerative brain diseases.

Eftud2-deficient microglia exhibit enhanced proliferation

A recent study has shown that Eftud2 is also expressed in neural crest cells (Beauchamp et al., 2021). To further explore the physiological role of Eftud2 in microglia, we used the microglia-specific inducible CX3CR1-CreER mouse strain, which allows for manipulation of gene expression specifically in microglia (Yona et al., 2013). Because this mouse strain requires tamoxifen to induce Cre recombinase, we first detected the activity and function of tamoxifen-induced Cre recombinase. By crossing a CX3CR1-CreER mouse with an Ai9 mouse, we generated CX3CR1-CreER; Ai9 reporter mice. To verify the effectiveness of Cre recombinase, we performed immunofluorescence staining on cryosections of CX3CR1-CreER; Ai9 mouse brain tissue using Iba-1, a marker for microglia in the CNS (Additional Figure 2A ^{(1.4MB, tif)} ; Li et al., 2020). In the absence of tamoxifen, there was little production of td-Tomato⁺ cells in the brains of CX3CR1-CreER; Ai9 mice (Additional Figure 2B ^{(1.4MB, tif)} ). In contrast, most td-Tomato⁺ cells were colocalized with Iba-1⁺ microglia after 5 days of 4-OH-TAM administration (P < 0.0001; Additional Figure 2B ^{(1.4MB, tif)} ). These results demonstrated that Cre-mediated recombination was highly effective in CX3CR1-CreER mice.

Next, we crossed Eftud2^f/f mice (Lv et al., 2019) with CX3CR1-CreER mice expressing tamoxifen-induced Cre recombinase to generate CX3CR1-CreER; Eftud2^f/f cKO mice. Furthermore, we injected 4-OH-TAM intraperitoneally at either P14 or P30 to obtain microglia-specific Eftud2 knockout mice. Injection of 4-OH-TAM led to the deletion of Eftud2 in microglia (P < 0.0001; Additional Figure 2C ^{(1.4MB, tif)} and D ^{(1.4MB, tif)} ), but not in GFAP⁺ astrocytes or NeuN⁺ neurons (Additional Figure 2E ^{(1.4MB, tif)} and F ^{(1.4MB, tif)} ). Then, we used 4-OH-TAM-injected CX3CR1-CreER; Eftud2^f/f mice as cKO mice and Eftud2^f/f mice as controls to examine whether the number of microglia varied in different brain regions after ablation of Eftud2 at P30 (Figure 2A). The number of Iba-1⁺ microglia was significantly higher in the hippocampus, prefrontal cortex, striatum, and cerebellum in the cKO mice compared with that in control littermates (prefrontal cortex, striatum, P < 0.05 or hippocampus, cerebellum, P < 0.001; Figure 2B).

Figure 2.

Eftud2-deficient microglia exhibit enhanced proliferation.

(A) Representative images of Iba-1 (green, Alexa Fluor 488) and DAPI immunostaining in hippocampus, striatum, prefrontal cortex and cerebellum of Eftud2^f/f and cKO mice after 4-OH-TAM injection. The number of Iba-1⁺ microglia was significantly higher in the hippocampus, prefrontal cortex, striatum, and cerebellum in the cKO mice compared with control littermates. Scale bar: 50 μm. (B) Iba-1-positive microglial cell count. (C) Iba-1 (green, Alexa Fluor 488) and Ki-67 (red, Alexa Fluor 568) immunostaining in cortex of Eftud2^f/f and cKO mice after 4-OH-TAM injection. Microglia proliferation rates were significantly higher in cKO mice compared with control mice. Scale bars: 20 μm. (D) Iba-1 and Ki-67 double-positive microglial cell count. Data are presented as mean ± SEM. At least three mice were used for each genotype. *P < 0.05, ***P < 0.001, ****P < 0.0001 (two-tailed unpaired Student’s t-test). 4-OH-TAM: 4-OH-Tamoxifen; cKO: conditional knockout; DAPI: 4',6-diamidino-2-phenylindole; Eftud2: elongation factor Tu GTP binding domain protein 2; Iba-1: ionized calcium-binding adaptor molecule 1.

The number of microglia in the brain is normally maintained in a relatively stable range by balancing proliferation and apoptosis (Askew et al., 2017). Because Eftud2 deletion increased the number of microglia, we next investigated whether the increased number of microglia in cKO mice was due to an increased proliferative capacity after loss of Eftud2. To test this hypothesis, we carried out immunofluorescence staining using anti-Ki67 antibody, which has been widely used as a marker of cell proliferation (Sobecki et al., 2016; Remnant et al., 2021). We observed a dramatically elevated number of Ki67⁺ cells colocalizing with Iba1⁺ cells in the brain of cKO mice after tamoxifen induction compared with the controls (Figure 2C). To further investigate the effect of Eftud2 deletion on microglia proliferation, we quantified microglia proliferation by counting the number of Ki67⁺Iba1⁺ double-positive cells. Microglia proliferation rates were significantly higher in cKO mice than in control mice (P < 0.0001; Figure 2D).

Previous studies have shown that the increase in microglia number is concentrated in the first 3 weeks after birth (Nikodemova et al., 2015; Lv et al., 2019). To examine the effect of Eftud2-specific knockout on developing microglia, 4-OH-TAM treatment was administrated to cKO mice at P14. We found that the number of microglia in different brain regions of cKO mice was significantly elevated compared with that in controls at P21 (striatum, P < 0.05, hippocampus, prefrontal cortex, P < 0.01 or cerebellum, P < 0.001; Additional Figure 3A ^{(1,019KB, tif)} and B ^{(1,019KB, tif)} ). Accordingly, many more Ki67⁺Iba1⁺ double-positive cells were observed in cKO mice than in control littermates (P < 0.0001; Additional Figure 3C ^{(1,019KB, tif)} and D ^{(1,019KB, tif)} ).

Therefore, we determined that the absence of Eftud2 resulted in an increase in the number of microglia in both developing and adult mouse brains, and this effect was observed in multiple brain regions. Although many studies have shown regional heterogeneity in microglia in the brain (Grabert et al., 2016; De Biase and Bonci, 2019), our results showed that after Eftud2 deficiency in microglia, there was a similar increase in the number of microglia in various brain regions. These results may indicate that Eftud2 has a common proliferative effect in microglia in multiple brain regions.

Eftud2-deficient microglia show morphological transformation into amoeboid cell shape

The morphology of microglia is closely related to its function (Perry et al., 2010; Morrison and Filosa, 2013). To better understand the role of Eftud2 in microglia, we next evaluated microglial morphology in different brain regions, including the hippocampus, prefrontal cortex, striatum and cerebellum, using Imaris software (Figure 3A). Eftud2-deficient microglia showed enlarged cell bodies (prefrontal cortex, striatum, P < 0.05 or hippocampus, cerebellum, P < 0.001) accompanied by decreased branch numbers (cerebellum, P < 0.05, hippocampus, prefrontal cortex, striatum, P < 0.001), terminal points (cerebellum, P < 0.01, hippocampus, prefrontal cortex, P < 0.001, striatum, P < 0.0001), and reduced total branch length (hippocampus, striatum, prefrontal cortex, cerebellum, P < 0.01) than in control mice (Figure 3B–E). Consistent with previous results, the morphological changes of microglia were similar across the multiple brain regions (Figure 3A). Moreover, we observed the same morphological changes in Eftud2-depleted developmental microglia at P14, including enlarged cell bodies (prefrontal cortex, P < 0.05; striatum, P < 0.001; hippocampus and cerebellum, P < 0.0001), decreased branch numbers (hippocampus, prefrontal cortex, and cerebellum, P < 0.001; striatum, P < 0.0001), terminal points (hippocampus, prefrontal cortex, and cerebellum, P < 0.001; striatum, P < 0.0001), and reduced total branch length (striatum, P < 0.01; hippocampus, prefrontal cortex, and cerebellum, P < 0.001) than in control mice (Additional Figure 4A ^{(3.6MB, tif)} –E ^{(3.6MB, tif)} ). These results further suggest that despite the heterogeneity of microglia in different brain regions, Eftud2 may have a common function in microglia throughout the brain. Taken together, our results suggest that Eftud2 deletion in microglia leads to dysregulation of cell proliferation and morphological transformation with amoeboid cell body and short branches.

Figure 3.

Eftud2-deficient microglia show morphological transformation.

(A) Imaris-based 3D reconstruction images of Iba-1⁺ microglia of Eftud2^f/f and cKO mice at P42. Eftud2-deficient microglia showed enlarged cell bodies accompanied by decreased branch points, terminal points, and reduced total branch length. Scale bars: 10 μm. (B–E) Imaris-based morphometric analysis of microglia of Eftud2^f/f and cKO mice at P42 after 4-OH-TAM induction. Data are presented as mean ± SEM. At least three mice were used for each genotype. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed unpaired Student’s t-test). 3D: Three-dimensional; 4-OH-TAM: 4-OH-tamoxifen; cKO: conditional knockout; Eftud2: elongation factor Tu GTP binding domain protein 2; Iba-1: ionized calcium-binding adaptor molecule 1; P42: postnatal day 42.

Eftud2-deficiency promotes the activation of anti-inflammatory phenotype of microglia

Activated microglia are typically divided into two states: M1 (also known as classically activated) with a proinflammatory phenotype, and M2 (known as alternatively activated) with an anti-inflammatory phenotype (Cherry et al., 2014; Ronaldson and Davis, 2020). Therefore, we used iNOS and Arg1, markers of the proinflammatory and anti-inflammatory phenotypes, respectively (Tang and Le, 2016), costained with Iba-1 to determine the subtype of activated microglia (Figure 4A–D). Eftud2 cKO mice had a significantly elevated number of Iba-1⁺Arg1⁺ cells (P < 0.0001), but not Iba-1⁺iNOS⁺ cells (P > 0.05), compared with controls (Figure 4B–E). These results indicated that Eftud2 ablation induced the activation and transformation of microglia into the anti-inflammatory phenotype instead of the proinflammatory phenotype. To further identify the phenotype of the increased population of Ki67⁺Iba1⁺ microglial cells in Eftud2 cKO mice, iNOS and Arg1 were costained with both Iba1 and Ki67 (Figure 4A–D). Ki67⁺Iba1⁺ staining was mainly present in Arg1⁺ cells (Ki67⁺Iba1⁺Arg1⁺, P < 0.0001), whereas there was little or no expression in iNOS⁺ cells (Ki67⁺Iba1⁺iNOS⁺, P > 0.05; Figure 4C–F). Taken together, our data indicated that Eftud2 deficiency specifically enhanced the proliferation of the anti-inflammatory phenotype microglia.

Figure 4.

Eftud2 deficiency promotes activation of anti-inflammatory phenotype microglia.

(A) Iba-1 (green, Alexa Fluor 488), Ki-67 (red, Alexa Fluor568), and iNOS (blue, Alexa Fluor 405) triple immunostaining in cortex of Eftud2^f/f and cKO mice after 4-OH-TAM injection. Iba-1⁺iNOS⁺ and Iba-1⁺iNOS⁺Ki67⁺ cells in Eftud2 cKO mice were not significantly different from controls. Scale bars: 20 μm. (B) Iba-1 and iNOS double-positive microglial cell count. (C) Iba-1, Ki-67 and iNOS triple-positive microglial cell count. (D) Iba-1 (green, Alexa Fluor 488), Ki-67 (red, Alexa Fluor568), and iNOS (blue, Alexa Fluor 405) triple immunostaining in cortex of Eftud2^f/f and cKO mice after TAM injection. Iba-1⁺Arg1⁺ and Iba-1⁺Arg1⁺Ki67⁺ cells in Eftud2 cKO mice were significantly increased compared with those in controls. Scale bars: 20 μm. (E) Iba-1 and Arg1 double-positive microglial cell count. (F) Iba-1, Ki-67, and Arg1 triple-positive microglial cell count. Data are presented as mean ± SEM. At least three mice were used for each genotype. ****P < 0.0001 (two-tailed unpaired Student’s t-test). 4-OH-TAM: 4-OH-Tamoxifen; Arg1: arginase-1; cKO: conditional knockout; Eftud2: elongation factor Tu GTP binding domain protein 2; Iba-1: ionized calcium-binding adaptor molecule 1; iNOS: inducible nitric oxide synthase; ns: not significant.

In addition, we knocked down Eftud2 in BV2 microglia with siRNA specifically targeting Eftud2 (P < 0.0001, compared with controls; Figure 5A). Transcription of inflammatory cytokines and microglia phenotypic markers were detected by RT-PCR in control and Eftud2 knockdown BV2 cells. Compared with the control, Eftud2 knockdown did not significantly alter the expression of iNOS and TNFα (P > 0.05; Figure 5B and C). In contrast, the mRNA levels of Arg1 and IL-10 were significantly upregulated in Eftud2 knockdown microglia compared with controls (P < 0.05 and P < 0.001, respectively; Figure 5D and E). We also assessed the phagocytic activity of microglia after Eftud2 knockdown by fluorescent microspheres (Figure 5F). Eftud2 knockdown microglia engulfed more beads than control microglia (number of beads, P < 0.0001 or phagocytic efficiency, P < 0.001; Figure 5G and H). This indicated that Eftud2 knockdown microglia had stronger phagocytic activity. Taken together, our data suggested that Eftud2 deletion promoted the activation of anti-inflammatory phenotype microglia.

Figure 5.

Eftud2 knockdown promotes anti-inflammatory phenotype activation in BV2 cells

(A) RT-PCR analysis of Eftud2 expression in BV2 cells infected with siRNA against Eftud2 (SiEftud2). (B–E) RT-PCR analysis of iNOS, TNF-α, Arg1, and IL-10 mRNA levels normalized to negative control (NC) after infection with SiEftud2. (F) Fluorescent microspheres (red) and DAPI (blue) in BV2 cells after infection with SiEftud2. Eftud2-knockout microglia engulfed more beads than controls. Scale bars: 5 μm. (G, H) Quantification of microglial phagocytosis. Data are presented as mean ± SEM. At least three mice were used for each genotype. *P < 0.05, ***P < 0.001, ****P < 0.0001 (two-tailed unpaired Student’s t-test). Arg1: Arginase-1; DAPI: 4',6-diamidino-2-phenylindole; Eftud2: elongation factor Tu GTP binding domain protein 2; Iba-1: ionized calcium-binding adaptor molecule 1; IL-10: interleukin-10; iNOS: inducible nitric oxide synthase; NC: negative control; RT-PCR: real-time polymerase chain reaction; siEftud2: short interfering RNA against Eftud2; TNF-α: tumor necrosis factor-α; ns: not significant.

Eftud2 regulates microglial phenotype activation via the NF-κB pathway

Many studies have demonstrated that M2 phenotype activation of microglia is protective (Ahmed et al., 2017; Ji et al., 2019). Given that Eftud2 deficiency promoted anti-inflammatory phenotype activation of microglia, we speculated that Eftud2 deletion in microglia may suppress inflammatory responses by promoting anti-inflammatory phenotype activation. To investigate this, we used LPS to stimulate BV2 microglia for 0, 12, and 24 hours, respectively. RT-PCR analysis showed that the expression of iNOS and TNFα in Eftud2 knockdown microglia was significantly suppressed after LPS stimulation, compared with control (12 hours, P < 0.01 or 24 hours, P < 0.001; Figure 6A and B). Moreover, we also found that knockdown of Eftud2 significantly increased the expression levels of Arg1 (24 hours, P < 0.001 or 0, 12 hours, P < 0.0001) and IL-10 under LPS stimulation (12 hours, P < 0.01, 12 hours, 24 hours, P < 0.001; Figure 6C and D). In addition, we observed that LPS-stimulated macrophage phagocytosis of fluorescent microspheres was significantly enhanced in Eftud2-knockdown BV2 microglial cells, which had a higher number of fluorescent microspheres within the soma when compared with controls (P < 0.01; Figure 6E–G). This suggested that Eftud2 may be involved in LPS-induced inflammatory responses by regulating the activation of proinflammatory and anti-inflammatory phenotypes.

Figure 6.

Eftud2 regulates proinflammatory and anti-inflammatory phenotype activation of microglia involved in inflammatory responses via the NF-κB pathway.

(A–D) RT-PCR analysis of iNOS, TNFα, Arg1, and IL-10 mRNA levels in NC and SiEftud2 microglial cells after exposure to LPS for the indicated times. (E) Fluorescent microspheres (red) and DAPI (blue) in NC and SiEftud2 microglial cells after exposure to LPS. Eftud2-knockout microglia engulfed more beads than NC groups. Scale bar: 5 μm. (F, G) Quantification of microglial phagocytosis. (H) Immunoblots of TLR4, MyD88, p65, p-p65 and β-actin proteins in NC, and SiEftud2. (I) Quantification of TLR4, MyD88, p65, and p-p65 levels normalized to β-actin. (J) RT-PCR analysis of TLR4, MyD88, and p65 mRNA levels in NC and SiEftud2 microglial cells after exposure to LPS. Data are presented as mean ± SEM. At least three independent experiments were repeated. **P < 0.01, ***P < 0.001, ****P < 0.0001 (two-tailed unpaired Student’s t-test). Arg1: Arginase-1; DAPI: 4',6-diamidino-2-phenylindole; Eftud2: elongation factor Tu GTP binding domain protein 2; Iba-1: ionized calcium-binding adaptor molecule 1; IL-10: interleukin-10; iNOS: inducible nitric oxide synthase; LPS: lipopolysaccharide; MyD88: myeloid differentiation primary response gene 88; NC: negative control; NF-κB: nuclear transcription factor-κB; ns: not significant; RT-PCR: real-time polymerase chain reaction; siEftud2: short interfering RNA against Eftud2; TLR4: Toll-like receptor 4; TNF-α: tumor necrosis factor-α.

Next, we investigated the possible signaling mechanism by which Eftud2 regulates microglial activation in the inflammatory response. The TLR4/NF-κB pathway has been shown to be involved in polarization of the microglia M1/M2 phenotypes (Zhang et al., 2019). Previous studies have also shown that LPS activates NF-κB signaling (Bode et al., 2012; Zhou et al., 2020). To determine whether the TLR4/NF-κB signaling pathway was affected in Eftud2-deficient BV2 cells, we used western blot assay to detect the expression of several important NF-κB signalsomes, including TLR4, MyD88, and total and p-p65, in Eftud2-deficient and control cell lysates. Eftud2 knockdown significantly decreased the expression of MyD88 and p-p65 compared with control cells (P < 0.01), and the expression of total p65 and TLR4 was not affected in Eftud2 deficient cells (P > 0.05; Figure 6H and I). Notably, we also found downregulation of MyD88 (P < 0.01) and no significant changes in TLR4 and p65 (P > 0.05) at the mRNA level (Figure 6J). Taken together, these results indicated that suppression of NF-κB signaling pathway activation is involved in Eftud2 deficiency-induced phenotypic activation of microglia in inflammatory responses.

Discussion

This study indicated an important role of Eftud2 in maintaining microglia homeostasis. Importantly, our findings suggest that Eftud2 regulates microglial proinflammatory and anti-inflammatory phenotype activation involved in inflammatory responses through the NF-κB pathway. Previous findings have confirmed that Eftud2, an important component of the spliceosome, plays an anti-inflammatory and antiviral role by modulating AS of immune factors (De Arras et al., 2014; Lv et al., 2019). However, its role in microglia has not been reported. Therefore, our results extend previous work by demonstrating the function of Eftud2 in microglia.

Eftud2 mutations were initially found in mandibulofacial dysostosis type Guion-Almeida patients (Lines et al., 2012) and have been frequently reported in recent years (Lehalle et al., 2014, 2015; Huang et al., 2016; Yu et al., 2018; Thomas et al., 2020). More recently, a comparative genomics study identified Eftud2 as an innate immunomodulator (De Arras et al., 2014). In addition, Eftud2 has been shown to be upregulated in both colitis model mice (Lv et al., 2019) and hepatocellular carcinoma patients (Tu et al., 2020). These results indicate that Eftud2 plays an important role in inflammation and tumorigenesis. In this study, we found that Eftud2 expression was dramatically elevated in the brain of AD mice. Microglial proliferation and activation concentrated around amyloid plaques in the brain is a prominent feature of AD, and chronic stimulation of microglia by Aβ may be detrimental and lead to long-term inflammation (Heneka et al., 2015; Colonna and Butovsky, 2017). Therefore, this result suggests that Eftud2 may play a key role in microglial activation in AD. We also observed that Eftud2 was persistently overexpressed in microglia 24 hours after LPS stimulation, further confirming the critical role of Eftud2 in inflammation. In zebrafish mutants, mutations in Eftud2 lead to abnormal proliferation and p53-dependent apoptosis of neural progenitor cells (Lei et al., 2017). Interestingly, specific deletion of Eftud2 in myeloid-like cells (Lv et al., 2019) and neural crest cells (Beauchamp et al., 2021) did not result in an abnormal proliferative phenotype. In our study, the proliferation of Eftud2-deficient microglia was significantly increased compared with control microglia, which was represented by an increase in microglia number. This may indicate that Eftud2 plays different roles in different cell types.

Microglia are activated immediately once they encounter foreign or harmful substances (Cherry et al., 2014). We found that Eftud2-deficient microglia showed an amoeba-like activation pattern. Proinflammatory phenotype activation is indicated by the increased release of proinflammatory factors (TNF-α, IL-6, and iNOS) (Qin et al., 2016; Tang and Le, 2016), and anti-inflammatory phenotype activation is indicated by increased release of anti-inflammatory factors (IL-10, CD209, and Arg1) (Tang and Le, 2016; Zhou et al., 2020). We showed that after Eftud2 deletion, the markers of Arg1⁺ anti-inflammatory phenotype microglia were significantly increased, whereas iNOS⁺ proinflammatory phenotype microglia were not significantly changed. Furthermore, we determined that both the abnormally proliferating microglia and the activated microglia were the anti-inflammatory phenotype. To support our conclusion, we used siRNA to knock down Eftud2 in BV2 microglia. Consistent with our in vivo results, we found that Eftud2 knockdown upregulated the expression of anti-inflammatory phenotype markers IL-10 and Arg1, and the expression of iNOS was not significantly changed. This suggests that Eftud2 deletion in microglia promotes microglia anti-inflammatory phenotype activation. More interestingly, in our study, Eftud2 knockdown in BV2 microglia increased the uptake of fluorescent microspheres. We speculate that Eftud2 knockdown may promote phagocytosis by microglia by inducing the anti-inflammatory phenotype.

Several transcriptional regulators have been identified to be involved in pro- and anti-inflammatory polarization of microglia, including the activator protein 1 family (AP-1), NF-κB family, and STAT1 (Holtman et al., 2017). Furthermore, specific deletion of Eftud2 in myeloid cells prevents colitis by inhibiting NF-κB activation (Lv et al., 2019). In this study, we found that Eftud2 knockdown in microglia attenuated LPS-induced expression of MyD88 and p-p65, indicating that the splice disorders may be responsible for inhibition of the NF-кB signaling pathway. However, in this study we did not determine whether Eftud2 deletion impairs NF-кB pathway activation via splicing changes of other molecules, which will need further detailed molecular characterization and investigation.

Notably, microglia have recently emerged as key players in the pathogenesis of AD, but the mechanisms of how they participate in AD progression remain unclear. Human genetic evidence suggests that microglia have a protective function in AD (Hansen et al., 2018). However, there is also substantial evidence that activated microglia can exacerbate neuronal damage in AD brains (Hansen et al., 2018). In particular, microglia can respond to protein aggregation and dying neurons in a proinflammatory manner, thereby causing damage to neurons by releasing inflammatory mediators. In this study, we found that the expression of Eftud2 was significantly increased in 5xFAD AD mouse brains. Interestingly, our data also showed that specific deletion of Eftud2 in microglia led to activation of anti-inflammatory microglia but inhibition of the proinflammatory response. Therefore, our results provide a potential intervention target to ameliorate AD pathogenesis by downregulation of Eftud2 expression specifically in microglial cells. More detailed research is needed to support this hypothesis in future work.

In conclusion, our study suggests that Eftud2 is essential for the maintenance of microglia polarization and homeostasis in both physiological, neural degenerative, and LPS-induced pathological states. In particular, our findings indicate that Eftud2 regulates microglial proinflammatory and anti-inflammatory phenotype activation involved in inflammatory responses through the NF-κB pathway. However, in this study, we did not explore the effect of Eftud2 deletion in microglia on mouse function, especially in AD mice. It is worth noting that anti-inflammatory phenotype activation of microglia has long been considered to be a potential mechanism for its neuroprotective effect. Therefore, Eftud2 may serve as a novel therapeutic target for spinal cord injury, stroke, AD and other CNS diseases.

Additional files:

Additional Figure 1 ^{(1.1MB, tif)} : Flowchart of the experiment.

Additional Figure 1

Flowchart of the experiment.

(A) Schematic diagram of generating inducible microglia Eftud2 conditional knockout mice. 4-OH-TAM induces CXCR1-CreER recombinase to produce microglia-specific knockout of Eftud2. (B) Experimental schedule for P14 4-OH-TAM injection. 4-OH-TAM was injected at P14, and the injection was continued for 5 days, and collected at P21. (C) Experimental schedule for P30 4-OH-TAM injection. 4-OH-TAM was injected at P30, and the injection was continued for 5 days, and collected at P42. 4-OH-TAM: 4-OH-Tamoxifen; P: postnatal day.

Additional Figure 2 ^{(1.4MB, tif)} : Conditional knockout of Eftud2 specifically in microglia in CX3CR1-CreER; Eftud2^f/f cKO mice.

Additional Figure 2

Conditional knockout of Eftud2 specifically in microglia in CX3CR1-CreER; Eftud2f/f cKO mice.

(A) Iba-1 (green, Alexa Fluor 488) and td-Tomato (red) immunostaining in cortex from CX3CR1-CreER; Ai9 reporter mice with or without 4-OH-TAM induction, respectively. In the absence of tamoxifen, there was little production of td-Tomato+ cells in the brains of CX3CR1-CreER; Ai9 mice. In contrast, most td-Tomato+ cells were co-located with Iba-1 + microglia after 5 days of 4-OH-TAM administration. Scale bars: 20 μm. (B) Quantification of Iba-1 and td-Tomato-double-positive microglial cells with or without 4-OH-TAM induction. (C) Iba-1 (green, Alexa Fluor 488) and Eftud2 (red, Alexa Fluor 568) immunostaining in the prefrontal cortex of Eftud2f/f and cKO mice after 4-OH-TAM induction. Injection of 4-OH-TAM led to the deletion of Eftud2 in microglia. Scale bars: 5 μm. (D) Quantification of Iba-1 and Eftud2-double-positive microglial cells in indicated genotypes. (E) Immunofluorescent staining of GFAP (green, Alexa Fluor 488) and Eftud2 (red, Alexa Fluor 568) in the striatum of Eftud2f/f and cKO mice after 4-OH-TAM induction. Eftud2 expression in astrocytes was unaffected after tamoxifen injection. Scale bars: 20 μm. (F) Immunofluorescent staining of NeuN (green, Alexa Fluor 488) and Eftud2 (red, Alexa Fluor 568) in the cortex of Eftud2f/f and cKO mice after 4-OH-TAM induction. Eftud2 expression in neurons was unaffected after tamoxifen injection. Scale bars: 20 μm. Data are expressed as mean ± SEM. At least three mice were used for each genotype. ****P < 0.0001 (two-tailed unpaired Student’s t-test). 4-OH-TAM: 4-OH-Tamoxifen; cKO: conditional knockout; DAPI: 4’,6-diamidino-2-phenylindole; Eftud2: elongation factor Tu GTP binding domain protein 2; GFAP: glial fibrillary acidic protein; Iba-1: ionized calcium-binding adaptor molecule 1.

Additional Figure 3 ^{(1,019KB, tif)} : Ablation of Eftud2 in microglia promotes their proliferation during development.

Additional Figure 3

Ablation of Eftud2 in microglia promotes their proliferation during development.

(A) Immunofluorescent staining of Iba-1 (green, Alexa Fluor 488) and DAPI (blue) in hippocampus, striatum, prefrontal cortex and cerebellum of Eftud2flox/flox and cKO mice at P21 after 4-OH-TAM induction. Representative images are shown. The number of microglia in different brain regions of cKO mice were significantly increased compared to controls at P21. Scale bar: 50 μm. (B) Quantification of the density of Iba-1+ microglial cells in the indicated brain regions. (C) Immunofluorescent staining of Iba-1 (green, Alexa Fluor 488) and Ki67 (red, Alexa Fluor 568) in prefrontal cortex of Eftud2flox/flox and cKO mice after 4-OH-TAM induction. More Ki67+Iba1+ double-positive cells were observed in cKO mice compared to control littermates. Scale bar: 20 μm. (D) Quantification of the ratio of Iba-1+Ki67+ to Iba-1+ cells between Eftud2flox/flox and cKO mice at P21 after 4-OH-TAM induction. Data are presented as the mean ± SEM. At least three mice were used for each genotype. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (two-tailed unpaired Student’s t-test). 4-OH-TAM: 4-OH-Tamoxifen; cKO: conditional knockout; DAPI: 4',6-diamidino-2-phenylindole; Eftud2: elongation factor Tu GTP binding domain protein 2; Iba-1: ionized calcium-binding adaptor molecule 1; P21: postnatal day 21.

Additional Figure 4 ^{(3.6MB, tif)} : Eftud2 deficiency in the developmental microglia show morphological transformation.

Additional Figure 4

Eftud2 deficiency in the developmental microglia show morphological transformation.

(A) Imaris-based three-dimensional reconstruction images of Iba-1+ microglia of Eftud2flox/flox and cKO mice at P21. Scale bars: 10 μm. Eftud2-deficient microglia showed enlarged cell body accompanied by decreased branch points, terminal points, and reduced total branch length. (B-E) Imaris-based morphometric analysis of microglia of Eftud2flox/flox and cKO mice after injection of TAM at P21. Data are presented as the mean ± SEM. At least three mice were used for each genotype. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (two-tailed unpaired Student’s t-test). 4-OH-TAM: 4-OH-Tamoxifen; cKO: conditional knockout; Eftud2: elongation factor Tu GTP binding domain protein 2; P21: postnatal day 21.

Additional file 1: Open peer review report 1 ^{(81.8KB, pdf)} .