Intact sociability in mice with elevated neuronal translation

We generated a conditional eIF4E overexpression allele at the Rosa26 locus (R26Eif4e) that expresses Myc-tagged eIF4E in a Cre-dependent manner (Supplementary Fig. 1a, b). eIF4E-Myc interacted with eIF4G as efficiently as untagged eIF4E (Supplementary Fig. 1c), indicating that the Myc tag does not interfere with the function of eIF4E. As it is generally believed that elevated protein synthesis in neurons causes ASD-like behaviors27, we first overexpressed eIF4E in neurons by crossing R26Eif4e mice with Syn1-Cre mice, which selectively express Cre in neurons as early as embryonic day 12.529, to generate Syn1-Cre;R26Eif4e/Eif4e mice (termed NN4E mice thereafter) (Fig. 1a). Levels of total eIF4E (eIF4E + eIF4E-Myc) in the NN4E hippocampus were more than twice as high as those in control mice (Fig. 1b). Interestingly, neuronal transgenic eIF4E expression significantly reduced endogenous eIF4E levels (Fig. 1b and Supplementary Fig. 2a). In fact, we found that one copy of the R26Eif4e allele was not sufficient to significantly increase levels of total eIF4E in the brain due to this negative feedback regulation (1.01 ± 0.10 for Syn1-Cre;R26Eif4e/+ vs. 1.00 ± 0.09 for R26Eif4e/+, p = 0.92, n = 5 mice per genotype). Using surface sensing of translation (SUnSET) which measures incorporation of puromycin into nascent polypeptides30, we found that protein synthesis rate was doubled in cultured NN4E hippocampal neurons over control neurons (Fig. 1c). These results indicate that neuronal protein synthesis is increased in NN4E mice.

Fig. 1: Neuronal eIF4E overexpression elevates anxiety without altering social interaction. a Syn1-Cre;R26Eif4e/Eif4e (NN4E) mice overexpress eIF4E in neurons, while R26Eif4e/Eif4e mice serve as controls (Ctrl). b Levels of eIF4E in hippocampal extracts prepared from control and NN4E mice. The eIF4E immunoblot reveals both endogenous eIF4E (lower band) and overexpressed eIF4E-Myc (upper band). Alpha tubulin was used as a loading control. n = 3 per genotype. **p = 0.0015 by two-sided t-test. c Protein synthesis in cultured hippocampal neurons as revealed by puromycin incorporation. n = 3 mice per genotype. **p = 0.0023 and *p = 0.015 by two-sided t-test. d Time spent in the central zone during the first 5 min in open field tests. Male: 15 control mice and 13 NN4E mice; Female: 15 control mice and 13 NN4E mice. **p = 0.0076, *p = 0.0218 by two-sided t-test. e Percentage of marbles buried. n = 15 control mice and 13 NN4E mice. *p = 0.0247 by two-sided t-test. f, g Sociability of male (f) and female (g) NN4E mice as revealed in three-chamber sociability tests. Male: 17 control mice and 14 NN4E mice; Female: 14 control mice and 13 NN4E mice. Two-way analysis of variance (ANOVA) with Fisher’s LSD post-hoc test: *p < 0.05, **p < 0.01, ***p < 0.001, and n.s. not significant. All data are shown as mean ± s.e.m. Source data are provided as a Source Data file. Full size image

We assessed if elevated protein synthesis in neurons leads to abnormal behaviors in mice. As revealed in rotarod and open-field tests (Supplementary Fig. 2b, c), NN4E mice had intact motor function. We examined anxiety-like behaviors by performing open field, elevated plus maze, and light-dark box tests. Anxious mice tend to avoid open, exposed, and brightly illuminated areas. Although NN4E and control mice spent comparable time in the light chamber in light-dark box tests (Supplementary Fig. 2e), NN4E mice of both sexes spent significantly less time in the center of the open field than control mice in the first 5 min of a 30-min open field test (Fig. 1d). In addition, male NN4E mice stayed longer in the closed arm of an elevated plus maze than control mice (Supplementary Fig. 2d). These results indicate that elevated neuronal protein synthesis increases anxiety. In agreement with the requirement of protein synthesis for long-term memory31, NN4E mice showed more freezing in the contextual chamber 24 h after fear condition training than control mice (Supplementary Fig. 2g). Conversely, working memory was unaffected in NN4E mice as measured by T-maze alternation (Supplementary Fig. 2f). We then ran marble burying and 3-chamber sociability tests to determine if elevated neuronal protein synthesis leads to repetitive behaviors and deficits in social interaction. Although female NN4E mice showed increased repetitive behaviors in marble burying tests relative to control mice (Fig. 1e), male NN4E mice were unaffected (Supplementary Fig. 2h). Unexpectedly, NN4E mice of both sexes stayed longer in the chamber with a holder holding a stranger mouse than in the chamber with an inanimate object (empty holder) and spent more time in investigating the stranger mouse than the empty holder, as did control mice in 3-chamber sociability tests (Fig. 1f, g). These behavioral results indicate that elevated neuronal protein synthesis does not lead to deficits in social interaction associated with ASD. This suggests that social interaction impairments observed in Eif4ebp2 knockout and transgenic βT-Eif4e mice16,17 are attributable to elevated protein synthesis in glial cells.

Elevated microglial translation leads to ASD-like behavior

We next overexpressed eIF4E in astrocytes (Supplementary Fig. 3a–c), which is the largest population of glia in the brain, using a Cre transgene driven by the astrocyte-specific promoter for glial fibrillary acidic protein (GFAP-Cre)32 which becomes active during late embryogenesis and the first postnatal week33. We found that astrocytic eIF4E overexpression did not alter repetitive and social behaviors in mice of either sex (Supplementary Fig. 3d–f). Thus, we shifted our efforts to microglia, which make up ~10% of brain cells34.

We employed Cx3cr1CreER mice25 to overexpress eIF4E in microglia. To determine the efficiency for tamoxifen to activate Cre recombinase in microglia, we crossed Cx3cr1CreER/+ mice to Rosa26Ai9/+ mice35 to generate Cx3cr1CreER/+;Rosa26Ai9/+ mice. A single tamoxifen injection (180 mg/kg, subcutaneously) was administered to Cx3cr1CreER/+;Rosa26Ai9/+ mice at P0. Tamoxifen activated CreER to excise the loxP-flanked transcription blocker of the Rosa26Ai9 allele in microglia, leading to tdTomato expression (Supplementary Fig. 4a). There is nearly 100% colocalization (n = 3 mice) between tdTomato and the microglial marker, ionized calcium binding adapter molecule 1 (Iba1), in the hippocampus and cortex (Supplementary Fig. 4b), indicating extremely high specificity in the expression of the Cx3cr1CreER gene. Furthermore, the recombination activity of the CreER protein is tightly controlled by tamoxifen, as we did not detect tdTomato expression in Cx3cr1CreER/+;Rosa26Ai9/+ mice in the absence of tamoxifen injection (Supplementary Fig. 4c). This observation indicates that a single tamoxifen injection in newborn pups is sufficient to activate Cre recombinase selectively in microglia of Cx3cr1CreER mice.

Newborn pups from R26Eif4e/Eif4e × Cx3cr1CreER/+;R26Eif4e/Eif4e crosses were treated with tamoxifen (180 mg/kg, subcutaneously) to generate control (R26Eif4e/Eif4e) and MG4E (Cx3cr1CreER/+;R26Eif4e/Eif4e) mice that should express eIF4E-Myc in microglia (Fig. 2a). Immunoblotting analysis of brain lysates revealed eIF4E-Myc expression in MG4E mice (Supplementary Fig. 4d). Immunohistochemistry revealed that eIF4E levels were lower in microglia than in neurons in control mice, but the transgenic overexpression increased microglial eIF4E levels more than two folds (Supplementary Fig. 5a). To further assess microglial eIF4E overexpression, we employed an immunopanning method36 to purify microglia from control and MG4E mice at P10 (Supplementary Fig. 6). Analysis of purified microglia revealed that two copies of Cre-excised R26Eif4e alleles increased levels of total eIF4E by 21–28% and protein synthesis rate by 28–35% in both male and female MG4E microglia compared to control microglia (Fig. 2b). Interestingly, eIF4E-Myc expression did not reduce endogenous eIF4E levels in microglia (Fig. 2b), as it did in neurons (Supplementary Fig. 2a) and astrocytes (Supplementary Fig. 3c). These results indicate that transgenic eIF4E-Myc expression elevates microglial protein synthesis to a similar extent in both sexes of mice.

Fig. 2: Overexpression of eIF4E in microglia leads to social deficits. a Genotypes of control (Ctrl) and MG4E mice. b Microglial eIF4E overexpression and protein synthesis in cultured microglia isolated from both sexes of control and MG4E mice. n = 3 per condition. Two-sided t-test: puromycin, female **p = 0.0099 and male *p = 0.0217; eIF4E, female **p = 0.0088 and male *p = 0.0293. c Time spent in the central zone during the first 5 min of open field tests. Male: 15 control mice and 14 MG4E mice; Female: 23 control mice and 25 MG4E mice. n.s. not significant by two-sided t-test. d, e Sociability of MG4E mice. Male: 14 control mice and 14 MG4E mice; Female: 23 control mice and 25 MG4E mice. Two-way ANOVA with Fisher’s LSD post-hoc test for chamber time and social investigation: *p < 0.05; **p < 0.01; ***p < 0.001; n.s. not significant. Two-sided t-test for social preference index: male, **p = 0.0067; female, p = 0.7440. f Social habituation test. n = 16 male control and 14 male MG4E mice. Two-way ANOVA with Fisher’s LSD post-hoc test: *p = 0.0159 between control and MG4E mice in trial 1; ###p < 0.001 between trial1 and trial4 in control mice; #p = 0.0334 between trial 4 and trial 5 in control mice; n.s. not significant for comparisons of trial 1 vs. trial 4 and trial 4 vs. novel in MG4E mice. g Habituation index. **p = 0.0013 by two-sided t-test. h, i Novel object recognition. n = 12 control mice and 12 MG4E mice. Two-way ANOVA with Fisher’s LSD post-hoc test: *p < 0.05 and n.s. not significant (h). Two-sided t-test: *p = 0.0297 (i). j, Self-grooming. The 30-min self-grooming test was divided into three 10-min segments. n = 6 mice per genotype. Two-way ANOVA with Fisher’s LSD post-hoc test was used for each 10-min segment, whereas two-sided t-test was used for the whole 30-min test period. *p = 0.0276; **p = 0.0016; n.s. not significant. Source data are provided as a Source Data file. Full size image

There is a difference in the extent of microglial eIF4E overexpression between the two aforementioned assays. To understand the discrepancy, we established cultures enriched for neurons, astrocytes, or microglia. To our surprise, eIF4E levels in cultured microglia were higher than those in cultured neurons (Supplementary Fig. 5b), although eIF4E levels are higher in neurons than in microglia in vivo on the basis of immunohistochemistry (Supplementary Fig. 5a). This result indicates that cell culturing somehow upregulates microglial eIF4E expression, which should cause underestimation of transgenic eIF4E expression. Therefore, it is likely that microglia in MG4E mice have more than twice as much eIF4E and protein synthesis as those in control mice, as do neurons in NN4E mice and astrocytes in AC4E mice.

Microglia are the most abundant CNS macrophages, which also include perivascular macrophages, meningeal macrophages and choroid-plexus macrophages37. In addition to CNS macrophages, CX3CR1 is expressed in peripheral monocytes and inflammatory macrophages25,37. In contrast to fast turnover of peripheral CX3CR1-expressing cells, CNS macrophages are long-living37. We ran behavioral tests when MG4E mice were at 2–3 months of age when the peripheral monocytes present at the time of tamoxifen injection should have been turned over, so that peripheral cells express little or no elF4E-Myc in tested mice (Supplementary Fig. 5c).

MG4E mice had normal locomotion in open field tests, indicating normal motor function (Supplementary Fig. 7a). They did not show anxiety-like behaviors in open field tests (Fig. 2c), elevated plus maze tests, or light-dark box tests (Supplementary Fig. 7b, c). Interestingly, MG4E mice displayed sexual dimorphism in 3-chamber sociability tests. Male MG4E mice displayed social interaction deficits, as they spent similar amounts of time investigating a stranger mouse and an inanimate object (Fig. 2d), whereas female MG4E mice had normal social interaction behaviors (Fig. 2e). To further characterize social behaviors of male MG4E mice, we performed social recognition tests in the home cage-like environment, an assay which relies on the mouse’s innate tendency to investigate a novel social partner and decrease the investigation of a known social stimulus (social habituation)38. Consistent with the social interaction deficit observed in 3-chamber sociability tests, male MG4E mice spent less time investigating a novel stimulus mouse in trial 1 (Fig. 2f). Furthermore, social habituation (trial 4 vs. trial 1) and dishabituation (novel stimulation mouse vs. trial 4) were impaired in male MG4E mice (Fig. 2f, g), indicating that their ability to adapt to novel social stimuli was attenuated, a phenomenon which has been linked to the pathognomonic social impairment and behavioral rigidity in ASD patients39. Again, female MG4E mice performed normally in social recognition tests (Supplementary Fig. 7f, g). These results indicate that elevated protein synthesis in microglia leads to social interaction deficits only in male mice.

We evaluated learning and memory in MG4E mice by conducting contextual fear conditioning and novel object recognition tests. Both sexes of control and MG4E mice showed similar long-term hippocampus-dependent contextual fear memory (Supplementary Fig. 7d). However, male, but not female, MG4E mice spent less time investigating a novel object than control littermates (Fig. 2h, i and Supplementary Fig. 7h), indicating recognition memory deficits in male MG4E mice. Thus, male MG4E mice exhibit selective deficits in cognitive functions.

To determine whether MG4E mice display repetitive behaviors, one of the core domains of ASD, we performed marble-burying and self-grooming tests. Although both sexes of MG4E mice performed normally in marble-burying tests (Supplementary Fig. 7e), male, but not female, MG4E mice spent significantly more time grooming than control mice (Fig. 2j and Supplementary Fig. 7i).

One copy of the Cx3cr1 gene is disrupted in Cx3cr1CreER/+ mice25. To rule out the possibility that the observed ASD-like phenotype in MG4E mice results from Cx3cr1 haploinsufficiency, we examined the behaviors of WT and Cx3cr1CreER/+ mice in three-chamber sociability tests (Supplementary Fig. 8a–d) and novel object recognition tests (Supplementary Fig. 8e–j). Both male and female Cx3cr1CreER/+ mice were normal in these behavioral tests.

Collectively, these results indicate that elevated protein synthesis in microglia leads to ASD-like phenotypes in mice, including male bias, deficits in social interaction, increased repetitive behaviors, and impaired cognitive functions.

Elevated protein synthesis alters microglial morphology

We next sought to investigate why microglial eIF4E overexpression increased protein synthesis in both sexes, but only caused ASD-like behaviors in males. We performed Iba1 immunohistochemistry to examine the impact of elevated protein synthesis on microglia in the medial prefrontal cortex (mPFC), hippocampus and striatum, the three brain regions that have been implicated in ASD pathophysiology40,41,42. We found that microglia in the mPFC were more numerous and larger in 2-week-old male MG4E mice than male control littermates (Fig. 3a–c). The same phenotype was observed in the hippocampus and striatum (Supplementary Fig. 9a–f). In contrast, elevated microglial protein synthesis did not affect the density and size of microglia in the mPFC, hippocampus and striatum of 2-week-old female MG4E mice (Fig. 3d–f and Supplementary Fig. 9g–l). Semi-automatic quantitative morphometric 3D measurements of microglia confirmed the increase in size and complexity of microglia in 2-week-old male MG4E mice (Fig. 3g). By 6 weeks of age, microglia in the mPFC, hippocampus and striatum in male MG4E mice remained larger than those of male control mice; however, their density became normal or slightly lower compared to control mice (Fig. 3h–j, n and Supplementary Fig. 10a–f). Again, elevated protein synthesis had no detectable effect on microglial size and density in 6-week-old female mice (Fig. 3k–m and Supplementary Fig. 10g–l). These data indicate that there is a sexual difference in microglial response to elevated protein synthesis.

Fig. 3: Iba1 immunohistochemistry reveals altered microglial density and morphology in male MG4E mice. a–c Increased microglial density and size in the mPFC of male MG4E mice. n = 7 control mice and 6 MG4E mice. Ten microglia from each mouse were randomly selected for measurement of cell size (cross section area). *p = 0.0342 and ***p < 0.001 by two-sided t-test. Scale bar, 100 μm. d–f Comparable microglial density and size in the mPFC between female control and MG4E mice. n = 6 control mice and 5 MG4E mice. Five to ten microglia from each mouse were randomly selected for measurement of cell size (cross section area). n.s. not significant by two-sided t-test. Scale bar, 100 μm. g Three-dimension reconstruction of microglia in the mPFC of 2-week-old male mice. n = 7 mice per group. Three to five microglia from each mouse were reconstructed for determination of cell size (volume) and number of processes. **p = 0.0037 and ***p = 0.0005 by two-sided t-test. Scale bar, 20 μm. h–m Microglia density and size in the mPFC of 6-week-old male (h–j) and female (k–m) MG4E mice. Ten microglia in each brain region of each mouse were randomly selected for measurement of cell size (cross section area). Male, n = 6 mice per genotype; female, n = 5 control mice and 6 MG4E mice. Two-sided t-test: ***p < 0.001 and n.s. not significant. Scale bar, 100 µm. n Three-dimension reconstruction of microglia in the mPFC of 6-week-old male mice. Four microglia from each mouse were reconstructed. Six control mice and five MG4E mice. *p = 0.0385 by two-sided t-test. Scale bar, 20 µm. All data are shown as mean ± s.e.m. Source data are provided as a Source Data file. Full size image

Inactivating mutations in the PTEN and FMR1 genes account for a large percentage of human syndromic ASD3. To determine if microglial changes observed in MG4E mice also occur in mice that model these two syndromes, we measured microglial density and size in 2-week-old male Pten+/− and Fmr1 knockout (KO) mice. Microglia in the mPFC, hippocampus and striatum were significantly larger in Pten+/− and Fmr1 KO mice than WT littermates (Fig. 4a, b and Supplementary Figs. 11a, 12a). Moreover, microglial density was increased in the Pten+/− mPFC and Fmr1 KO striatum (Fig. 4a and Supplementary Fig. 12a). The lack of elevated microglial density in all brain regions of 2-week-old male Pten+/− and Fmr1 KO mice could be due to a smaller degree of protein synthesis increase relative to MG4E mice. We notice that microglial density in tamoxifen-treated male R26Eif4e/Eif4e (control) mice is lower than that in untreated male WT mice at 2 weeks of age (compare Figs. 3c, 4a, b), which is possibly due to growth retardation of mice caused by tamoxifen injection at P0.

Fig. 4: Altered microglial density and size in Pten+/− and Fmr1 KO mice. a Microglial density and size in the mPFC of 2-week-old male and female Pten+/- mice. Male, n = 5 mice per genotype. Female, n = 6 mice per genotype. Five to ten microglia from each condition were randomly selected for measurement of cell size (cross section area). Two-sided t-test: male, cell density *p = 0.0317, cell size **p = 0.001; female, cell density p = 0.0725, cell size *p = 0.0232, n.s. not significant. Scale bar, 100 µm. b Microglial density and size in the mPFC of 2-week-old male and female Fmr1 KO mice. Male, n = 7 mice per genotype. Female, n = 4 WT mice and 5 KO mice. 5-10 microglia from each condition were randomly selected for measurement of cell size (cross section area). Two-sided t-test: **p = 0.0046, and n.s. not significant. Scale bar, 100 µm. c, d Microglial density and size in the mPFC of 6-week-old male Pten+/− (c) and Fmr1 KO (d) mice. c n = 5 mice per condition; d n = 6 WT mice and 5 Fmr1 KO mice. Five to ten microglia from each condition were randomly selected for measurement of cell size (cross section area). n.s., not significant by two-sided t-test. Scale bar, 100 μm. All data are shown as mean ± s.e.m. Source data are provided as a Source Data file. Full size image

The microglial morphological phenotype observed in 2-week-old male Pten+/− and Fmr1 KO mice also displays sexual dimorphism. While microglia in 2-week-old female Pten+/− mice show some morphological alterations, the phenotype is not as prominent as observed in male Pten+/− littermates. Compared to control mice, microglia in 2-week-old female Pten+/− mice were larger in the mPFC and the striatum but not in the hippocampus, and their density was not significantly altered in all the three brain regions (Fig. 4a and Supplementary Fig. 11b). Furthermore, the density and size of microglia was normal in 2-week-old female Fmr1 KO mice (Fig. 4b and Supplementary Fig. 12b). Similar to what was observed in MG4E mice, the morphological phenotype in male Pten+/− and Fmr1 KO mice is attenuated with age, so that the phenotype in these mice disappeared by 6 weeks of age (Fig. 4c, d and Supplementary Figs. 13, 14).

Synaptic alterations in male MG4E mice

As microglia play important roles in neuronal development, the observed changes in microglial number and morphology could alter synaptic development and function in male MG4E mice, which then leads to ASD-like behaviors. To investigate this possibility, we examined synaptic structures in layer 2 of the mPFC prelimbic area (PrL) in 6-week-old male control and MG4E mice using serial block-face scanning electron microscopy (Fig. 5a). Three-dimension reconstruction of dendrites revealed that male MG4E mice had higher spine density, with contributions from both non-synaptic and synaptic spines compared to control mice (Fig. 5b). Male MG4E mice had smaller spines and synapses on average (Fig. 5c). To examine synaptic maturation, we counted multiple-synapse spines (MSS) and multiple-synapse boutons (MSB), which are believed to be the structural basis of synaptic multiplicity26. Synaptic multiplicity increases in the hippocampus during postnatal development and is considered an indicator of synapse maturation43. We found that male control and MG4E mice had comparable MSS and MSB density in layer 2 of the PrL (Supplementary Fig. 15a, b), suggesting that reduced synapse size in male MG4E mice is not indicative of immaturity.

Fig. 5: Structural and functional alterations in synapses of MG4E mice. a Schematic diagram of mPFC serial block-face scanning electron microscopy (SB-SEM). b Spine density in the mPFC of 6-week-old male control and MG4E mice. Representative dendrites reconstructed from SB-SEM images show dendritic spines (gray) and presynaptic terminals (blue). Graphs show spine density, synaptic spine density and percentage of non-synaptic spines. 5 control mice, 40 dendrites and 1187 spines; 6 MG4E mice, 30 dendrites and 1120 spines. Two-sided t-test (spine density, **p = 0.0017; synaptic spine density, *p = 0.0112; non-synaptic spine *p = 0.0253). Scale bar, 2 µm. c Spine volume and synaptic size. Representative dendrites reconstructed from SB-SEM images show dendritic spines (blue) and postsynaptic density (PSD, light yellow). Graphs show PSD size and spine volume. 5 control mice, 40 dendrites and 1098 spines; 6 MG4E mice, 30 dendrites and 999 spines. Two-sided t test (PSD area, **p = 0.0044; spine volume, **p = 0.0037). Scale bar, 2 µm. d Density of asymmetric and symmetric synapses in the mPFC of 6-week-old male and female control and MG4E mice. Images show representative asymmetric and symmetric synapses (arrows). n = 3 mice and 30 images per mouse for each group. **p = 0.0059 by two-sided t-test. Scale bar, 2 µm. e Spine density in mPFC layer 5 neurons and hippocampal CA1 neurons of 2-week-old (2 W) and 6-week-old (6 W) male control and MG4E mice. 2 W, 6 mice per genotype; 6 W, 6 control mice and 7 MG4E mice; 2–5 neurons in each brain region per mouse. *p = 0.0362, **p = 0.0012 and ***p < 0.001 by two-sided t-test. Scale bar, 5 µm. f Levels of neuroligin 1 (NLGN1) and neuroligin 2 (NLGN2) in the mPFC and hippocampus. n = 4 control mice and 5 MG4E mice. Two-sided t test (mPFC, *p = 0.0409 for NLGN1 and *p = 0.0279 for NLGN2; hippocampus, *p = 0.0207 for NLGN1 and *p = 0.0229 for NLGN2). All data are shown as mean ± s.e.m. Source data are provided as a Source Data file. Full size image

Because dendritic spines are the postsynaptic sites for the vast majority of excitatory synapses44, higher spine density indicates more excitatory synapses. Indeed, transmission electron microscopy analysis revealed more asymmetric (excitatory) synapses in the PrL of 6-week-old male, but not female, MG4E mice compared to control mice (Fig. 5d). This analysis also found a normal density of symmetric (inhibitory) synapses in the PrL of both male and female MG4E mice (Fig. 5d). Thus, exaggerated protein synthesis in microglia increases the number of excitatory synapses in the mPFC in male mice.

Increased spine density has been observed in ASD patients45,46. To determine if spine density is also increased in other brain areas of male MG4E mice and at other ages, we employed the Thy1-GFP transgene47 to label isolated neurons in 2-week and 6-week-old mice. We found that male, but not female, MG4E mice had higher spine density in pyramidal neurons of mPFC layer 5 and hippocampal CA1 area at both ages (Fig. 5e and Supplementary Fig. 16a). These results indicate that elevated microglial protein synthesis increases the density of excitatory synapses in multiple brain areas.

We next examined whether levels of synaptic proteins are altered in the mPFC and hippocampus of MG4E mice. We found that adult male and female control and MG4E mice had comparable levels of presynaptic synaptophysin and postsynaptic neuroligin 3, neuroligin 4, PSD95 and GluA1 in the hippocampus and mPFC (Supplementary Figs. 15c, 15d, 16b, 16c). Neuroligins are cell-adhesion molecules important for synaptogenesis and have been implicated in ASD48. Levels of neuroligins 1–4 are increased in the hippocampus of Eif4ebp2 knockout and transgenic βT-Eif4e mice, which was interpreted as a result of increased mRNA translation in neurons16. Interestingly, levels of neuroligins 1 and 2 in the hippocampus and mPFC were significantly higher in male, but not female, MG4E mice, compared with sex-matched control mice (Fig. 5f and Supplementary Fig. 16b, c). This finding suggests that the observed increase in hippocampal levels of neuroligins in Eif4ebp2 knockout and transgenic βT-Eif4e mice could be a result of microglial alterations rather than increased eIF4E activity in neurons. This argument is supported by the observation that levels of neuroligins1–4 were comparable between male control mice and male NN4E mice that overexpress eIF4E in neurons (Supplementary Fig. 17). The increase in levels of neuroligins 1 and 2 is likely due to enhanced transcription in neurons, as levels of mRNAs for the two proteins show a trend of increase in male MG4E mice (Supplementary Fig. 15e).

To investigate if alterations in synaptic structure and proteins lead to abnormal synaptic function in male MG4E mice, we recorded miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) in layer 5 pyramidal neurons of the mPFC. The amplitude, but not the frequency, of mEPSCs was increased in male MG4E mice relative to control mice (Fig. 6a). In contrast, the amplitude of mIPSCs were slightly reduced in male MG4E mice, as indicated by a significant but small left shift of its cumulative curve (Fig. 6b). These results suggest an alteration in the excitation/inhibition (E/I) balance, which is believed to contribute to ASD3, in the mPFC. Indeed, we found that the normalized total charge transfer in male MG4E mice relative to control mice was significantly larger for mEPSCs than for mIPSCs (Fig. 6c). As expected on the basis of synapse density and levels of synaptic proteins, female control and MG4E mice had comparable mEPSCs, mIPSCs and charge transfer in the mPFC (Supplementary Fig. 16d, e).

Fig. 6: Abnormal synaptic function in male MG4E mice. a mEPSCs recorded in mPFC layer 5 neurons of male control and MG4E mice at 6–7 weeks of age. n = 20 cells from five control mice and 33 cells from 8 MG4E mice. Two-sided t-test for frequency and amplitude: **p = 0.0022; n.s. not significant. Two-sided Kolmogorov–Smirnov test for cumulative probability of mEPSC amplitude: ***p < 0.001. Scale bars, 50 pA (vertical) and 0.5 s (horizontal). b mIPSCs recorded in mPFC layer 5 neurons of male control and MG4E mice at 6–7 weeks of age. n = 17 cells from 4 control mice and 25 cells from 6 MG4E mice. Two-sided t-test for frequency (p = 0.6053) and amplitude (p = 0.1097): n.s. not significant. Two-sided Kolmogorov–Smirnov test for cumulative probability of mIPSC amplitude: ***p < 0.001. Scale bars, 25 pA (vertical) and 0.5 s (horizontal). c Relative changes in mEPSC (n = 33 cells) and mIPSC (n = 25 cells) total charge transfer in MG4E mice, normalized to control mice. p = 0.0453 by two-sided Kolmogorov–Smirnov test. All data are shown as mean ± s.e.m. Source data are provided as a Source Data file. Full size image

Collectively, our microscopic, biochemical and electrophysiological analyses reveal that elevated protein synthesis in microglia leads to synaptic alterations that are associated with ASD in male mice only, including increased spine density, increased levels of neuroligins and E/I imbalance. These synaptic changes likely contribute to deficits in social interaction, repetitive behaviors, and cognitive impairments in male MG4E mice.

Microglia are out of homeostatic state in male MG4E mice

To understand how microglial eIF4E overexpression alters synaptic structure and function, we analyzed hippocampal gene expression in male control and MG4E mice at 2 and 6 weeks of age using RNA-Seq. Weighted gene correlation network analysis (WGCNA)49 revealed 11 modules of highly co-expressed genes in control and MG4E mice (Supplementary Fig. 18a). Of note, the magenta module displayed a set of transcripts that were significantly upregulated in 2-week-old MG4E mice (Fig. 7a). Pathway enrichment analysis of genes within the magenta module revealed top KEGG pathways related to antimicrobial activities, including herpes simplex infection, antigen processing and presentation, phagosome, influenza A and autoimmune thyroid disease (Fig. 7b, c). Some of the disease-associated microglial genes found in neurodegenerative conditions50 were also upregulated in 2-week-old MG4E mice (28 out of 138 genes in the magenta module; Fig. 7c). By 6 weeks of age, the magenta module eigengene was no longer significantly upregulated in MG4E mice, suggesting that this set of transcripts were only upregulated during postnatal development. Interestingly, the herpes simplex virus infection and autoimmune thyroid disease pathways in the magenta module have been reported to be risk factors for ASD51,52,53. In addition to the upregulated genes (Supplementary Fig. 18b, c), we identified a group of downregulated genes by comparing 2-week-old male MG4E mice to control littermates (Fig. 7d and Supplementary Fig. 18b, d). Many of the downregulated genes (11/40, or 27.5%) are markers for homeostatic (M0) microglia54, including P2ry12, P2ry13, Cx3cr1, Tmem119, and Slco2b1 (Fig. 7e).

Fig. 7: Gene expression profiles in male control and MG4E mice. RNA-seq was performed using RNA samples isolated from hippocampi of 2-week-old (2 W) and 6-week-old (6 W) male control (Ctrl) and MG4E mice. a (top) Heatmap showing module-trait relationship of magenta module. Correlation coefficients between a module eigengene and trait (coded from −1 to 1) and corresponding p values (two-sided Student asymptotic p value for a given correlation was calculated using corPvalueStudent in WGCNA package) are shown in each cell. The color indicates the level of correlation. (bottom) Plot showing the expression of the module eigengene across sample categories. b Heatmaps of genes (138 genes) within magenta module. c Selected KEGG pathways and disease-associated microglial genes enriched in magenta module (logP values adjusted with Benjamini correction). d Heatmaps of downregulated genes (FDR < 0.05) in 2-week-old MG4E mice. (e Heatmaps of downregulated M0-homeostatic microglial genes in 2-week-old MG4E mice. f, g Heatmaps of top 40 M1 (f) and M2 (g) microglial genes in 2-week-old MG4E mice (Ctrl and MG4E, n = 4 mice). h, i RT-PCR validation of selected upregulated (h) and down-regulated (i) genes in hippocampi of 2-week-old male MG4E mice. n = 8 per genotype. *p < 0.05, **p < 0.01 and ***p < 0.001 by two-sided t-test. j RT–qPCR validation of selected genes in hippocampi of 6-week-old male MG4E mice. n = 7 control mice and 8 MG4E mice. n.s. not significant by two-sided t-test. All data are shown as mean ± s.e.m. Source data are provided as a Source Data file. Full size image

In addition to M0 state, microglia can achieve two other polarized states (M1 and M2) that are characterized by specific molecular signatures54,55. It is proposed that M1 microglia produce pro-inflammatory cytokines and M2 microglia are anti-inflammatory55. We found that very few M1 and M2 markers were upregulated in 2-week-old male MG4E mice (Fig. 7f, g). This indicates that elevated protein translation does not move microglia to M1 or M2 polarized states.

We validated the RNA-Seq results by measuring mRNA levels of a few select genes in 2-week and 6-week-old control and MG4E mice using quantitative RT-PCR (Fig. 7h–j). The RT-PCR analysis also found that the observed gene expression changes were absent in 2-week-old female MG4E mice (Supplementary Fig. 18e). Taken together, the RNA-Seq and RT-PCR analyses show that elevated protein synthesis shifts microglia from the homeostatic state to a functional state with properties that have been observed in neurodegenerative conditions.

Male MG4E microglia with altered phagocytosis and motility

Microglia are the primary phagocytes in the brain that engulf invading pathogens and cellular debris56. Upregulation of antimicrobial pathways including the phagosome pathway (Fig. 7c) prompted us to hypothesize that the phagocytic capacity of microglia is enhanced in 2-week-old male MG4E mice. Indeed, purified microglia from male MG4E mice phagocytized more than twice as many Aβ (1–42) aggregates as those from male control mice, but microglia from female control and MG4E mice had comparable phagocytosis (Fig. 8a).

Fig. 8: Microglial surveillance and synapse engulfment in MG4E mice. a Phagocytosis of FAM-Aβ (1–42) in cultured control and MG4E microglia. Male, n = 18 for control and n = 28 for MG4E; female, n = 14 for control and n = 13 for MG4E. **p = 0.0017 and n.s. not significant (p = 0.4919) by two-sided t-test. b, c Migration of microglia into FAM-Aβ (1–42) injection sites in 2-week-old male (b) and female (c) control and MG4E mice. Microglia clustering index is defined as (density of Iba1+ cells in the FAM-Aβ-covered area)/(density of Iba1+ cells in a contralateral site). Male, n = 6 control mice and 7 MG4E mice; female, n = 4 per genotype. Two-sided t-test: male mice, contralateral microglia density, **p = 0.0013; clustering index, **p = 0.0026, n.s. not significant. Scale bars, 100 μm. d Microglial surveillance in response to ATP treatment in male and female MG4E microglia at P14–P18. Microglial baseline motility was recorded for 5 min, then ATP was bath-applied to brain slices and recorded for another 10 min. Data was normalized to the mean process motility of first 5 min. Male, n = 15 microglia from 3 control mice and 20 microglia from 3 MG4E mice; female, n = 18 microglia from 3 control mice and 26 microglia from 3 MG4E mice. Male, two-way ANOVA for genotype during ATP treatment, F (1, 330) = 9.566, p = 0.002; female, two-way ANOVA for genotype during ATP treatment, F (1, 420) = 0.045, p = 0.831. Scale bar, 10 μm. e Engulfment of Homer1 by microglia. Upper panel shows confocal images of Homer1 and Iba1 double immunohistochemistry, and lower panel shows 3-D reconstruction of a microglial cell and Homer1 immunoreactivity. Arrowheads denote Homer1 inside the microglia. Male, 18 microglia from 6 control mice and 15 microglia from 5 MG4E mice; female, 15 microglia from 5 mice per genotype. *p = 0.0378 and n.s., not significant (p = 0.4891) by two-sided t-test. Scale bars, 5 μm. All data are shown as mean ± s.e.m. Source data are provided as a Source Data file. Full size image

Given that microglia have been shown to prune synapses through phagocytosis during postnatal development23,24, we were puzzled by how elevated spine density (Fig. 5b, e) and enhanced microglial phagocytosis (Fig. 8a) co-exist in male MG4E mice. We noticed that the downregulated genes are associated with leukocyte cell–cell adhesion, leukocyte migration, and cell–cell adhesion (Supplementary Fig. 18d). Thus, we examined migration of microglia to an injection site of Aβ (1–42) aggregates in 2-week-old male control and MG4E mice. We found that the number of microglia migrated to the Aβ (1–42) injection site was significantly reduced in male, but not female, MG4E mice relative to control mice (Fig. 8b, c). These results indicate that elevated protein synthesis impairs the motility of microglia in male mice.

Microglial processes display two modes of motility: constant extension and retraction to survey the brain (baseline motility) and extension to the source of attractants such as ATP released from a damaged site (induced motility)57. Because synapse pruning involves contacts of microglial processes with synapses58, it should be affected by the motility of microglial processes. As elevated protein synthesis impaired the motility of whole microglia, it could also affect the motility of microglial processes. We monitored the baseline motility and ATP-induced motility of microglial processes in organotypic hippocampal cultures established from P14-P18 control and MG4E mice harboring the CD68-EGFP transgene59 which is selectively expressed in microglia. Irrespective of sex, microglial processes in control and MG4E mice had comparable baseline processes motility (Supplementary Fig. 18f). However, ATP-induced motility was impaired in male, but not female, MG4E mice (Fig. 8d).

Synapses to be eliminated may release a factor to initiate their interactions with microglial processes, and thereby deficits in the induced motility in male MG4E mice would impair engulfment of synapses. We assessed synapse engulfment by measuring the immunoreactivity of postsynaptic protein Homer1 inside microglia. Three-D reconstruction of Iba1 and Homer1 immunoreactivities revealed that the volume of Homer1 immunoreactivity inside microglia was significantly reduced in male, but not female, MG4E mice relative to control mice (Fig. 8e).

Collectively, these results indicate that induced motility of microglia is impaired in male MG4E mice, which might lead to reduced microglial engulfment of synapses and subsequently increased density of excitatory synapses, despite of enhanced microglial phagocytosis capacity in these mice.