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Postnatal downregulation of Fmr1 in microglia promotes microglial reactivity and causes behavioural alterations in female mice

Molecular Autism volume 16, Article number: 17 (2025) Cite this article

Abstract

Background

Fragile X syndrome is caused by the loss of the Fmr1 gene expression. Deletion of Fmr1 in various neuronal and non-neuronal subpopulations in the brain of mice leads to cell-type-specific effects. Microglia, immune cells critical for the refinement of neuronal circuits during brain development, have been implicated in various neurodevelopmental disorders, including fragile X syndrome. However, it is unknown whether reduced Fmr1 expression in microglia leads to molecular and behavioral phenotypes.

Methods

We downregulated Fmr1 in microglia during early and late postnatal development and studied the effect on microglial morphology and distinct behaviours.

Results

Female, but not male, adult mice with downregulation of Fmr1 in microglia during early development exhibited reactive microglia and behavioral phenotypes, including enhanced self-grooming and alterations in social interaction. Downregulation of Fmr1 in microglia during late development induced a milder phenotype, characterized by impaired preference for social novelty without affecting microglia morphology.

Conclusions

The downregulation of Fmr1 and its encoded protein FMRP in microglia contributes to behavioural phenotypes in a sex-specific manner.

Background

Fragile X syndrome (FXS) is a genetic neurodevelopmental disorder and the leading monogenic cause of intellectual disability and autism [1]. In FXS, expansion of the CGG repeat within the promoter region of the Fmr1 gene on the X chromosome leads to the transcriptional silencing of the Fmr1 gene and the subsequent absence of its protein product, fragile X messenger ribonucleoprotein 1 (FMRP) [2]. FMRP is an RNA-binding protein that regulates protein synthesis, mRNA stability, and trafficking [3]. Fmr1 knockout (KO) mice, which have been widely used to study FXS, exhibit numerous phenotypes reminiscent of FXS, including impaired social interaction and repetitive behaviors [4, 5].

Emerging evidence indicates that FMRP regulates molecular and behavioural phenotypes in a cell-type-specific manner. Deletion of Fmr1 in forebrain excitatory neurons results in enhanced locomotor activity, anxiety-like behavior, and amplified resting EEG gamma power [6, 7]. Deletion of Fmr1 in VGlut2-expressing glutamatergic neurons induces audiogenic seizures [8]. Fmr1 deletion in subpopulations of inhibitory neurons revealed that mice lacking Fmr1 in parvalbumin-expressing interneurons exhibit dysregulated de novo protein synthesis, anxiety-like behavior, and impaired social behavior, whereas mice lacking Fmr1 in somatostatin-expressing neurons show no changes in protein synthesis or behavior [9]. Deletion of Fmr1 in cerebellar Purkinje cells results in enhanced long-term depression (LTD) at the parallel fiber synapses, and deficits in delay eyeblink conditioning [10].

Apart from neurons, Fmr1 is also expressed in non-neuronal cells, such as astrocytes [11, 12] and microglia [13]. Selective ablation of Fmr1 in astrocytes decreased glutamate transporter GLT1 levels and increased neuronal sensitivity to extracellular glutamate [14]. Microglia are resident immune cells in the brain that play important roles in brain homeostasis, synaptic pruning, and the formation of neuronal circuits during early postnatal brain development [15,16,17]. Dysregulation of microglial function, including alterations in their activation state and abundance, is observed in postmortem brains of individuals with ASD [18] and Fmr1 KO mice [19]. Despite the crucial role of microglia in brain development and their involvement in distinct neurodevelopmental disorders [20,21,22], the role of microglial FMRP in FXS remains unknown. In this study, we downregulated Fmr1 in microglia during early and late postnatal development in both sexes and assessed behavioural and microglial phenotypes.

Methods

Animals

To generate mice with microglia-specific deletion of Fmr1, Fmr1fl/fl female mice [23] (kindly provided by Dr. David L. Nelson, Baylor College of Medicine) were crossed with male mice expressing Cre recombinase under the microglia-specific promotor, TMEM119CreERT2 (JAX, stock #031820) [24]. The Fmr1fl/y TMEM119CreERT2 mice were consequently crossed with Fmr1+/fl TMEM119CreERT2 mice to obtain female Fmr1fl/fl TMEM119CreERT2 mice. TMEM119CreERT2 mice were used as a control group. Mice were housed by genotype. Both experimental and control mice were injected with 4-OHT or tamoxifen, see “Pharmacological reagents” for details. All the mouse strains were maintained on the C57BL/6 background. Adult male and female mice (8-week-old) were used in all experiments. All experiments were conducted and analyzed by a researcher blind to the genotypes and treatments. The animals were housed in standard Plexiglas mouse cages with food and water available ad libitum and maintained on a 12-hour light/dark cycle with lights on at 7:00 AM. All procedures were compliant with the Canadian Council on Animal Care guidelines and approved by McGill University’s Animal Care Committee.

Immunohistochemistry

Mice were transcardially perfused with 1X PBS followed by 4% paraformaldehyde (PFA), pH 7.4. Following extraction, the brains were postfixed in PFA 4% for 24 h at room temperature. The fixed brains were then sectioned transversely using a vibratome to acquire 50-µm-thick sections. After washing with PBS, sections were blocked using 10% normal goat serum (NGS) and 0.5% Triton-X100 in PBS for two hours. The blocking was then followed by an overnight incubation with primary antibodies. For confirmation of FMRP downregulation, FMRP (1:200, Cat No. ab17722, Abcam) and Iba1 (1:500, Cat No. 234–308, Synaptic Systems) were used, and for 3-D image analysis, Iba1 (1:500, Cat No. 019-19741, Wako) and CD68 (1:500, Cat No. MCA1957, Bio-rad) were used in 0.5% Triton-X100 in PBS (PBS-T). After three washes with PBS, sections were incubated for two hours at room temperature with the corresponding secondary antibodies (1:500) diluted in PBS. DAPI (1:5000) was added to the solution in the last wash. Finally, sections were mounted on glass slides, imaged using a confocal microscope (Zeiss LSM 880) with a 63X/1.40 Oil DIC f/ELYRA objective, and images were analyzed using ImageJ (NIH). All images were captured using Z-stack mode with 15–20 optical sections/stack. The integrated density signal within the cell body of Iba1-positive cells in the corpus callosum was quantified using ImageJ on maximum intensity projection images. Two sections per animal were imaged, and the integrated density of FMRP in 5–10 microglia per image was quantified. The values of the total microglia were averaged to obtain a single value per mouse. 3D image analysis of microglia was performed using IMARIS (See IMARIS section for more details).

Open field test

Mice were individually placed into an illuminated (~ 1,200 lx) white non-porous plastic square box without bedding (50 cm x 50 cm x 31 cm) and given 10 min to freely explore the apparatus. Their activities were recorded and analyzed using the Noldus EthoVision XT video tracking software. The time spent inside and outside the defined zones (inner zone 33 cm x 33 cm) was then analyzed.

Marble burying test

After a 30-minute habituation period, mice were individually placed in a plexiglass cage (50 cm x 50 cm x 31 cm) filled with 5 cm of bedding. The mice were allowed to bury 18 shiny and clean marbles, which were equally distributed in three rows of 6 marbles, for 30 min. The marble was considered buried if at least 2/3 of it was covered by bedding. The limitation of this analysis is that it does not distinguish whether the marble is buried due to direct burying behaviours or walking/sitting on them in passing.

Self-grooming test

Mice were placed in a mouse cage with fresh bedding and allowed to habituate and explore the cage for 20 min. Grooming behavior during the last 10 min was recorded using an overhead rigged camera. The total time spent on self-grooming, and the number of grooming bouts were scored and analyzed.

Elevated plus maze

To assess anxiety-like behavior, mice were individually placed in the center of the elevated plus maze and allowed to explore for 5 min while their activity was recorded using a mounted camera positioned above the maze. The maze consisted of two open arms (30 cm × 5 cm) and two closed arms (30 cm × 5 cm × 15 cm), elevated 50 cm above ground level. The time spent in the open and closed arms, as well as the total number of arm entries, were calculated and analyzed.

Three-chamber social interaction test

A Plexiglass box with three interconnected compartments (36 cm x 28 cm x 30 cm) was used to assess social interaction and novelty-seeking behavior. In the first 10-minute habituation phase, the test mouse was placed in the middle compartment and was allowed to explore empty small wire cages located in the right and left compartments for 10 min. During the 10-minute sociability phase, the test mouse was exposed to an empty wire cage in one compartment and a wire cage containing a stranger mouse (Stranger 1, age- and sex-matched wild-type mouse, C57BL/6J) in the other compartment. In the final 10-minute novelty-seeking phase, the test mouse was allowed to explore the wire cage containing the now familiar Stranger 1, and a novel stranger (Stranger 2) confined in the other wire cage. All three phases began with placing the test mouse in the middle compartment and opening doorways to the right and left compartments. The left/right sides of the apparatus were counterbalanced to contain stimuli or empty wire cage across subjects. The light intensity in the room was 255 lx. The entire 30-minute test was recorded using an overhead-mounted camera. The total time spent in each compartment, the time spent in social interaction, and the total number of compartment entries were scored and analyzed. Social interaction time was defined as the time when the test mouse approached the empty or occupied wire cage at a distance of less than 1 cm.

Reciprocal (direct) social interaction test

After habituating mice for five minutes in a novel standard mouse housing cage (27 cm x 15 cm x 12 cm) with fresh corn cob bedding, mice were individually exposed to an unfamiliar age and sex-matched mouse (WT) for 10 min. The recorded video was then analyzed to measure the total time of interaction between the test mouse and the unfamiliar WT mouse (defined as nose-to-anogenital sniffing, nose-to-nose sniffing, and social grooming). The light intensity in the room was 255 lx.

Pharmacological reagents

4-OHT: 4-Hydroxytamoxifen (4-OHT) was first dissolved at 20 mg/ml in ethanol and then further diluted in sunflower oil to obtain a final concentration of 2 mg/ml with 10% ethanol. Mice then received intraperitoneal injections of 4-OHT (Sigma, Cat. No. H7904) once daily for five consecutive days from postnatal day 1 to postnatal day 5, with each animal receiving a total dosage of 0.25 mg. Tamoxifen: Tamoxifen (Sigma T5648) was dissolved in 10% ethanol and 90% sunflower oil to obtain 20 mg/ml concentration. Animals then were administered 100 mg/kg of tamoxifen or vehicle intraperitoneally daily for 5 consecutive days starting at postnatal day 30.

IMARIS

To study the morphology and phagocytic activity of microglia, Sholl and CD68 lysosome analyses of microglia were conducted on the hippocampal and cortical sections using IMARIS (9.7.2). Microglia and lysosomal volume were analyzed using Iba1 and CD68 staining, respectively. Thick Sect. (70 μm) of the hippocampus and cortex were used. High-resolution images of individual microglia using a Zeiss LSM 880 upright confocal microscope using a 63X oil objective (NA: 1.4) (38–48 μm z-stacks; xy resolution: 0.10 μm; z step: 0.2 μm, 1024 × 1024 px) were captured. The CZI file format (.czi) was converted into an Imaris file format (.ims) using a file converter, so it could be opened in Imaris 9.7.1. The Filament Tracer Module in IMARIS was used to reconstruct microglia for Sholl analysis, with the Iba1 channel being selected for this purpose. After reconstruction, the filament number of Sholl intersections data were generated by IMARIS and compiled into an Excel spreadsheet for further analysis. Lysosomal volume was measured by 3D reconstruction and overlapping CD68 signal with Iba1. The surface rendering model was utilized to identify microglia and CD68 surfaces for conducting volume-based analysis. Surfaces were reconstructed for CD68 and Iba1 separately. The amount of CD68 volume that colocalizes with the Iba1 signal was analyzed by IMARIS and combined in an Excel spreadsheet before analysis. Two sections per animal were imaged and analyzed. A total of 5–10 cells per section were analyzed. Each group consisted of five mice. IMARIS 3D reconstruction was utilized in conjunction with representative images of Iba1/CD68 to demonstrate the localization of CD68 within microglia.

Statistical analysis

Statistical analysis was conducted using GraphPad Prism 9 (GraphPad Prism Software Inc., USA) and the data were tested for normality using the Shapiro–Wilk test. The data are presented as mean ± standard error of the mean (s.e.m.), and significance was defined as p < 0.05. Differences between two groups were assessed using a two-tailed unpaired Student’s t-test or a nonparametric two-tailed Mann–Whitney test. Multiple group comparisons were performed using either one-way ANOVA or two-way ANOVA, followed by Tukey’s or Bonferroni’s post-tests. All behavioral assessments were scored and analyzed by an experimenter blinded to both genotypes and treatments. Each data point in the graphs corresponds to the number of animals.

Results

Microglia-specific downregulation of Fmr1

To delete Fmr1 in microglia, we crossed Fmr1fl/fl mice with mice expressing Cre recombinase under the control of the microglia-specific promoter, Tmem119 (TMEM119CreERT2). Male and female TMEM119CreERT2 mice were used as controls. To target early postnatal development, the experimental and control groups received 4-Hydroxytamoxifen (4-OHT, daily) during postnatal days 1 to 5 (mice referred to as Fmr1 cKOearly, Fig. 1A). For late postnatal development, experimental and control mice were treated daily with tamoxifen from day 30 to 34 (referred to as Fmr1 cKOlate, Fig. 1B). Previous work has reported that FMRP is detected in microglia in all examined brain areas during the first postnatal weeks; however, its expression is dramatically reduced in adult mice, particularly in the hippocampus and cerebellum [13]. In contrast, the expression of FMRP in microglia remains stable in the corpus callosum across the lifespan [13]. To confirm the downregulation of Fmr1 in microglia in our cKO mouse models, we measured FMRP expression in corpus callosum microglia of 8-week-old mice using immunohistochemistry with FMRP-specific antibodies and a microglial marker, Iba1. FMRP levels were significantly decreased in microglia of Fmr1 cKOearly (Fig. 1C and D) and Fmr1 cKOlate (Fig. 1E and F) mice.

Fig. 1
figure 1

Downregulation of the Fmr1 gene in microglia. (A, B) The schematics illustrate the early and late developmental downregulation of the Fmr1 gene induced by either 4-OHT (50 mg/kg/i.p.) or tamoxifen (100 mg/kg/i.p.) administration, respectively. (C-F) Immunostaining against FMRP in microglia (Iba1+ cells) shows ablation of FMRP in microglia at early and late postnatal developmental stages in the corpus callosum of Fmr1 cKOearly and Fmr1 cKOlate compared to the controls (C, Female: Fmr1 cKOearly vs. control, t8 = 4.60, **p = 0.0018; D, Male: Fmr1 cKOearly vs. control, t8 = 2.81, *p = 0.0227, n = 5 for all groups) and Fmr1 cKOlate (E, Female: Fmr1 cKOlate vs. control, t7 = 3.54, **p = 0.0094; F, Male: Fmr1 cKOlate vs. control, t8 = 3.07, *p = 0.0154), n = 4–5). ImageJ was used to quantify the FMRP integrated density signal within the cell body of Iba1-positive cells in the corpus callosum, using maximum intensity projection images. Two sections per animal were imaged, and the integrated density of FMRP in 5–10 microglia per image was quantified. The values were averaged to obtain a single value per mouse. Unpaired two-tailed Student’s t-test. Data points represent individual mice. Data are shown as mean ± SEM. The scale bars are 25 μm. *p < 0.05 and **p < 0.01

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Early development downregulation of microglial Fmr1 causes female-specific behavioural alterations and changes in microglial morphology

To study the role of Fmr1 in microglia during the early developmental stages in the pathogenesis of behaviors reminiscent of fragile X syndrome, adult Fmr1 cKOearly mice underwent behavioural testing. Female Fmr1 cKOearly mice spent less time in the inner zone and more time in the outer zone of the open field test compared to controls (Fig. 2B). Female Fmr1 cKOearly mice also exhibited increased self-grooming time (Fig. 2C) and marble-burying behaviors (Fig. 2D). No anxiety phenotype was observed in the elevated plus maze (Fig. 2E), as no differences were found in the time spent in the open arm or closed arm between the Fmr1 cKOearly and control mice (although a trend was observed). Female Fmr1 cKOearly mice showed deficits in the novelty-seeking phase of the three-chamber social interaction tests (Fig. 2F, right). Fmr1 cKOearly female mice also showed reduced direct social interaction in the reciprocal social interaction test, although it did not reach statistical significance (p = 0.0704, Supplemental Fig. 1A). Surprisingly, in contrast to females, male Fmr1 cKOearly mice exhibited no significant deficits in the open field, self-grooming, marble burying, elevated plus maze, and three-chamber social interaction tests (Fig. 2G-K; integrated male and female analyses are shown in Supplemental Fig. 1B-D). These findings indicate that the early postnatal downregulation of Fmr1 in microglia in female mice is sufficient to induce enhanced self-grooming and alterations in the novelty phase of the three-chamber social interaction test.

Fig. 2
figure 2

Early developmental downregulation of microglial Fmr1 causes sex-specific behavioural alterations (A) Schematic illustration of early deletion of Fmr1 gene in microglia. (B– F) Behavioral analysis of female Fmr1 cKOearly (KO n = 7, Control n = 10) showed that the mice spend less time in the inner zone and more time in the outer zone of the open field test (B, inner zone time: t15 = 2.20, *p = 0.0438 and outer zone time: t15 = 2.20, *p = 0.043). Female Fmr1 cKOearly mice spent more time self-grooming (C, t15 = 2.44, *p = 0.0273 ) and buried more marbles (D, **p = 0.0068). No anxiety-like behavior was observed in the elevated plus maze (E, ns, p > 0.05). In the three-chamber social interaction test, female Fmr1 cKOearly mice exhibited impaired novelty seeking (F, left: ns, p > 0.05), whereas no impairment was observed in the sociability phase of the test (F, right: t30 = 5.67, ****p < 0.0001). (G– K) No significant differences in male Fmr1 cKOearly mice as compared to controls in the open field, self-grooming, marble burying, elevated plus maze, and three-chamber social interaction tests (G– K, ns, p > 0.05 for all tests, n = 8–9). Unpaired two-tailed Student’s t-test was used for B-C, E, and G-I, and two-tailed Mann–Whitney test was used for D and J. The three-chamber social interaction test was analyzed using two-way ANOVA followed by Bonferroni’s multiple comparisons test. Data points represent individual mice. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001, ns– not significant

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We next studied whether the early postnatal downregulation of Fmr1 in microglia affects the morphology and phagocytic capacity of microglia. The analysis of microglial morphology using 3D reconstruction of Iba1 immunostaining showed that hippocampal and cortical microglia in female Fmr1 cKOearly mice exhibit diminished arborization, as indicated by the reduced number of intersections in 3D Sholl analysis (Fig. 3B, cortex; F, hippocampus), as well as the decreased branch number (Fig. 3C, cortex; G, hippocampus) and processes length (Fig. 3D, cortex; H, hippocampus). Moreover, volumetric analysis of CD68+ signal, indicative of microglial phagocytic capacity, showed a significant increase in CD68/Iba1 volume ratio in the cortex (Fig. 3E) and hippocampus (Fig. 3I) of female Fmr1 cKOearly mice. Conversely, male Fmr1 cKOearly mice exhibited no change in microglia morphology and CD68 volume (Fig. 3K-R). Decreased arborization of microglial processes combined with increased CD68 signal in Fmr1 cKOearly female mice suggests that the early postnatal downregulation of Fmr1 in microglia induces the transition of microglia to a reactive state in a sex-dependent manner [25].

Fig. 3
figure 3

Early developmental downregulation of microglial Fmr1 causes morphological changes and enhances phagocytic activity. (A) Representative images, skeleton, and volumetric 3D reconstructions of microglia. (B– I) 3D analysis of Iba1+ microglia revealed that female Fmr1 cKOearly microglia exhibit diminished arborization, as indicated by reduced number of intersections in 3D Sholl analysis (B, cortex: F12, 96 = 2.52, **p = 0.0062 and F, hippocampus: F12, 96 = 14.90, ****p < 0.0001), along with decreased branch number (C, cortex: t8 = 2.90, *p = 0.0198 and G, hippocampus: t8 = 3.12, *p = 0.0142) and processes length (D, cortex: t8 = 2.04, ns, p > 0.074 and H, hippocampus: t8 = 3.40, **p = 0.0093). Volumetric analysis of CD68 signal in microglia (Iba1+) showed a significant increase in CD68+/Iba1+ volume ratio in the cortex (E, t8 = 3.11, *p = 0.0143) and hippocampus (I, t8 = 2.78, *p = 0.0237) of female Fmr1 cKOearly mice. Conversely, male Fmr1 cKOearly mice exhibited no change in microglia morphology (representative reconstruction (J), Sholl analysis (K, O), branch number (L, P), and processes length (M, Q)) and phagocytosis capacity (N, R) in the cortex or hippocampus (Fig. 3J– R, ns, p > 0.05). Unpaired two-tailed Student’s t-test was used for branch number, processes length, and CD68. For Sholl analysis, two-way ANOVA followed by Bonferroni’s multiple comparisons test was used. The scale bars in A and J are 10 μm. n = 4–5 mice/group. Data points represent individual mice. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001, ns– not significant

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Fmr1 ablation in microglia during late development leads to deficits in the novelty-seeking phase of the three-chamber social interaction tests in female mice

We next tested mice with downregulation of Fmr1 in microglia during the late stage of development (Fmr1 cKOlate). Female Fmr1 cKOlate mice showed impairment in the novelty-seeking phase of the three-chamber social interaction test (Fig. 4F, right), but no deficits in the sociability phase (Fig. 4F, left), direct social interaction (Supplemental Fig. 1E), open field (Fig. 4B), self-grooming (Fig. 4C), marble burying (Fig. 4D), and elevated plus maze (Fig. 4E) compared to controls. No behavioral alterations were observed in male Fmr1 cKOlate mice (Fig. 4G-K). Notably, late-development downregulation of Fmr1 in microglia did not affect their reactivity state, as indicated by no alterations in microglial arborization in the cortex and hippocampus of male and female mice, as well as unchanged CD68+ volume (Supplemental Fig. 2, A-R). Thus, downregulation of microglial Fmr1 during the late postnatal stage has no major effect on microglial phenotypes and repetitive behaviours, but it causes deficits in the novelty-seeking phase of the three-chamber social interaction test in female mice.

Fig. 4
figure 4

Microglia-specific downregulation of Fmr1 during late development leads to a deficit in novelty-seeking phase of the three-chamber social interaction test in female mice. (A) The schematic illustrates the late downregulation of the Fmr1 gene in microglia. (B– F) The behavioral analysis of female Fmr1 cKOlate mice (n = 9) revealed impairment during novelty-seeking phase of the three-chamber social interaction test (F, right, ns, p > 0.05 ), but not during the sociability phase of the same test (F, left, t36 = 10.31, ****p < 0.0001). The female Fmr1 cKOlate mice behaviors in the open field, self-grooming, marble burying, and elevated plus tests were comparable to the controls (B– E, ns, p > 0.05). The male Fmr1 cKOlate mice did not show behavioral deficit during the open field, self-grooming, marble burying, and elevated plus tests in comparison to controls (G– J, ns, p > 0.05). The male Fmr1 cKOlate mice did not exhibit impairment in sociability or novelty-seeking phases of the three-chamber social interaction test (K, ****p < 0.0001 and ***p < 0.0001 for empty cage (E) vs. stranger 1 (S1) and stranger 2 (S2) vs. stranger 1 (S1) in control and male Fmr1 cKOlate). Unpaired two-tailed Student’s t-test was used for D, and G-J. Two-tailed Mann–Whitney test was used for B, C, and E. The three-chamber social interaction test was analyzed using two-way ANOVA followed by Bonferroni’s multiple comparisons test. Data points represent individual mice. Data are shown as mean ± SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001, ns– not significant

Full size image

Discussion

We found that decreasing Fmr1 in microglia during the early postnatal development is sufficient to induce morphological changes in microglia, along with enhanced self-grooming and deficits in the novelty-seeking phase of the three-chamber social interaction test in adult female, but not male mice. Downregulation of Fmr1 in microglia during the later developmental stage led to impairments in the novelty-seeking phase of the three-chamber social interaction test, but not self-grooming behavior or microglial morphology. Thus, we identified a critical postnatal developmental window for FMRP in microglia, linked to sexually dimorphic cellular and behavioral phenotypes.

FMRP expression in microglia across various brain regions peaks around postnatal day 10 and declines thereafter [13]. The stronger effect of early postnatal downregulation is consistent with the peak of FMRP expression in microglia and the critical period (postnatal days 7–14 [26, 27] when microglia shape developing neuronal circuits through synaptic pruning and neurogenesis [28, 29]. Our results are consistent with previous studies linking microglia and FXS [12, 30]. Specifically, Fmr1 KO mice display lower microglial density [31] and elevated reactivity of microglia to lipopolysaccharide, indicated by exaggerated phagocytosis and pro-inflammatory responses [19]. Our findings are also consistent with emerging evidence linking microglia to autism, including alterations in their number, morphology [17], and the expression of microglia-specific genes [32, 33] in postmortem autistic brains, abnormalities in social behavior in mice with impaired microglia-mediated synaptic pruning, such as Cx3cr1 KO [34], and the induction of behavioural phenotypes reminiscent of autism by maternal immune activation [35, 36].

Microglia exhibit sexual dimorphism in various physiological processes. Overexpression of eIF4E in microglia elevated mRNA translation in both sexes, but induced social deficits and increased microglial density and size in male mice only [37]. Conversely, single-cell RNA sequencing of microglia in mouse models with high and normal anxiety-related behavior (HAB and NAB, respectively) revealed higher expression of genes associated with phagocytosis and synaptic engulfment in female HAB mice compared to males [38]. Accordingly, a greater amount of VGlut1+ excitatory synapses was engulfed by the female HAB microglia compared to the male microglia. Minocycline administration reduced synapse engulfment and anxiety-like behaviors in female but not male HAB mice 38). Female-specific effects of Fmr1 downregulation in microglia is an intriguing observation that requires further investigation. There are several potential explanations for the observed sex differences. First, the number and characteristics of microglia vary between males and females in a region-specific and age-dependent manner [39]. Downregulation of Fmr1 could differentially affect various brain regions, and accordingly, impact neuronal processes underlying complex behaviors in males and females. Second, the X chromosome contains a significant number of immune-related genes [39], raising the possibility that in the absence of Fmr1, X-linked immune-related genes may become dysregulated, preferentially affecting microglial homeostasis and function in females. Sex-specific effects might also be related to the interaction with sex hormones [40, 41] or alternatively, to the expression of TMEM119CreERT2 and the subsequent Fmr1 downregulation in only a fraction of microglia [24, 42], raising the possibility that microglia that do not undergo recombination compensate better in males than in females.

Limitations

This study investigated the effect of Fmr1 downregulation in microglia on microglial morphology and distinct behaviors which are altered in Fmr1 KO mice. Importantly, assessment of behaviors reminiscent of neurodevelopmental disorders, including autism, in mice is challenging [43] and requires additional tests to characterize alterations in sensory functions (vision, olfaction, touch), memory, and novelty detection [44]. We confirmed the downregulation of microglial FMRP expression in the corpus callosum, but not in other brain areas. This is because FMRP expression is substantially reduced to very low/undetectable levels in most brain regions by postnatal week 3, but remains readily detectable in the corpus callosum [13]. The TMEM119CreERT2 mouse line is highly specific to microglia, without leakage of Cre activity [24]. Finally, we assessed microglia morphology and CD68 expression in the cortex and hippocampus. It will be important to examine other brain areas and additional behaviors that are affected in neurodevelopmental disorders.

Conclusion

This study found that Fmr1 downregulation in microglia promotes microglial reactivity, enhances self-grooming behaviors and induces deficits in the novelty-seeking phase of the three-chamber social interaction test in female, but not male, mice. The exact mechanism by which FMRP-deficient microglia cause behavioral phenotypes remains unknown. While we assessed key phenotypes observed in Fmr1 KO mice, other phenotypes, such as audiogenic seizures, dendritic spine morphology and density, LTD, protein synthesis, and the activity of different signaling cascades, should be investigated in future studies.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

FXS:

Fragile X syndrome

FMRP:

Fragile X messenger ribonucleoprotein

VGlut2:

Vesicular-glutamate transporter 2

LTD:

Long-term depression

GLT1:

Glutamate transporter-1

ASD:

Autism spectrum disorder

4-OHT:

4-Hydroxytamoxifen

CD68:

Cluster of differentiation 68

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Acknowledgements

We thank Dr. David L. Nelson (Baylor College of Medicine, Houston) for kindly providing Fmr1fl/fl mice.

Funding

This work was supported by a grant from the Canadian Institutes of Health Research (CIHR, PJT-162412), the Azrieli Centre for Autism Research (ACAR) to A.K., and ERA-NET Neuron Sensory disorders project TRANSMECH (A.K. and C.G.G.). C.G.G. was supported by HFRI grant (Project No. 2556). M.H. was supported by the Brain Canada and Transforming Autism Care Consortium (TACC) graduate fellowships.

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Authors and Affiliations

  1. Department of Anesthesia, Faculty of Dental Medicine and Oral Health Sciences, McGill University, Montreal, QC, Canada

    Mehdi Hooshmandi, David Ho-Tieng, Kevin C. Lister, Weihua Cai, Calvin Wong, Nicole Brown, Jonathan Fan, Volodya Hovhannisyan, Sonali Uttam & Arkady Khoutorsky

  2. Department of Physiology, McIntyre Medical Sciences Building, McGill University, Montreal, QC, Canada

    Masha Prager-Khoutorsky

  3. Department of Biochemistry and Goodman Cancer Research Centre, McGill University, Montreal, Canada

    Nahum Sonenberg

  4. Biomedical Research Institute, Foundation for Research and Technology-Hellas, loannina, Greece

    Christos G. Gkogkas

  5. Alan Edwards Centre for Research on Pain, McGill University, Montreal, QC, Canada

    Arkady Khoutorsky

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  1. Mehdi Hooshmandi
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Contributions

M.H., C.G.G., and A.K. conceived the project, designed experiments, and supervised the research. M.H. performed immunohistochemistry and behavioral experiments, with the help of D.HT., K.C.L., W.C., C.W., N.B., S.U., and J.F. N.S., V.H., and M.P.K. assisted with data interpretation. M.H., C.G.G., and A.K. wrote the manuscript with input from all the authors.

Corresponding authors

Correspondence to Christos G. Gkogkas or Arkady Khoutorsky.

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All procedures were compliant with the Canadian Council on Animal Care guidelines and approved by McGill University’s Animal Care Committee.

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Supplementary Material 1

: Fig.1: Early developmental downregulation of microglial Fmr1 reduces reciprocal social interaction: (A) Reciprocal (direct) social interaction was assessed in Fmr1 cKOearly female mice. Unpaired two-tailed Student’s t-test was used for the analysis. (B-D) Behavioral results of Fmr1 cKOearly female and Fmr1 cKOearly male mice from open field inner zone (Genotype: F (1, 30) = 1.213; p = 0.2796, Sex: F (1, 30) = 3.281; p = 0.0801, Interaction: F (1, 30) = 2.887; p = 0.0960), outer zone (Genotype: F (1, 30) = 1.213; p = 0.2796, Sex: F (1, 30) = 3.281; p = 0.0801, Interaction: F (1, 30) = 2.887; p = 0.0960), self-grooming (Genotype: F (1, 30) = 0.06116; p = 0.8063, Sex: F (1, 30) = 0.1492; p = 0.7021, Interaction: F (1, 30) = 5.383; p = 0.0273*), and marble burying (F (1, 30) = 5.358; p = 0.0276*, F (1, 30) = 0.1853; p = 0.6699, F (1, 30) = 6.778; p = 0.0142*) tests were analysed together (analyses of each sex separately appear in Fig.2), using two-way ANOVA followed by Tukey’s multiple comparisons test. P values for significant post-hoc comparisons are indicated on the graphs. (E) Reciprocal (direct) social interaction was assessed in Fmr1 cKOlate female mice. Two-tailed Mann–Whitney test was used for the analysis. Data points represent individual mice. Data are shown as mean ± SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001, ns– not significant.

Supplementary Material 2

: Fig.2: The late developmental downregulation of Fmr1 in microglia does not affect their morphology or phagocytic activity: (A, J) Representative images, skeleton, and volumetric 3D reconstructions of microglia. (B– I) 3D and volumetric analysis of Iba1+ microglia revealed no alterations in microglial arborization in female and male Fmr1 cKOlate microglia as indicated by the unchanged number of 3D Sholl intersections, branch number, processes length, and no significant differences in phagocytic activity in cortex and hippocampus (ns, p > 0.05). Unpaired two-tailed Student’s t-test was used for branch number, processes length, and CD68. For Sholl analysis, a two-way ANOVA followed by Bonferroni’s multiple comparisons test was used. The scale bars in A and J are 10μm. n = 4–5 mice/group. Data points represent individual mice. Data are shown as mean ± SEM. ns– not significant.

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Hooshmandi, M., Ho-Tieng, D., Lister, K.C. et al. Postnatal downregulation of Fmr1 in microglia promotes microglial reactivity and causes behavioural alterations in female mice. Molecular Autism 16, 17 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13229-025-00648-2

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Keywords

Molecular Autism

ISSN: 2040-2392