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. 2026 Aug 20;16:605. doi: 10.1038/s41398-026-04360-4

A serotonergic mechanism is involved in the pro-cognitive effect of AM6545 treatment in a mouse model of fragile X syndrome

Lucía de los Reyes-Ramírez 1,2,#, Araceli Bergadà-Martínez 2,#, Sara Martínez-Torres 2, Irene Martínez-Gallego 3, Pilar Losada-Ruiz 3, Silvia Arboleya 4, Antonio González-Matute 3, Miguel Gueimonde 4, Rafael Maldonado 2,5, Antonio Rodríguez-Moreno 3, Andrés Ozaita 1,2,✉
PMCID: PMC13728186  PMID: 42971989

Abstract

Fragile X syndrome (FXS) is the principal monogenic syndrome leading to inherited intellectual disability and autism. It is caused by the silencing of FMR1 gene that leads to the loss in the expression of its encoded protein, the fragile X messenger ribonucleoprotein 1 (FMRP). In synapses, FMRP has a key role in local mRNA modulation to maintain synaptic plasticity. The Fmr1 KO (FX) mouse model shows cognitive impairment and some of the synaptic traits present in individuals with FXS, together with alterations in gut microbiota. Previous studies revealed that pharmacological and genetic cannabinoid type-1 receptor (CB1R) inhibition significantly prevented central key alterations in FX mice. Here, we aimed to evaluate the effect of a sub-chronic treatment with the largely peripherally-restricted CB1R antagonist AM6545. We found that AM6545 reduced memory deficits and restored enhanced hippocampal mGluR5-dependent long-term depression and aberrant dendritic spine density in FX mice. At the peripheral level, AM6545 modified altered FX mice fecal microbiota composition and intestinal Muc2 expression, while in the hippocampus AM6545 treatment upregulated hippocampal Htr4, the gene encoding for serotonin receptor 4 (5-HT4R). This upregulation, also observed in cecum, positively correlated with memory performance. Notably, acute pharmacological blockade of 5-HT4R abolished AM6545 pro-cognitive effect. Our results suggest that repeated peripheral CB1R inhibition ameliorates key central and peripheral alterations in FXS mouse model and modifies the expression of serotonergic receptors important for cognitive performance. Together, this evidence points to the peripheral targeting of CB1R as an approach to alleviate cognitive dysfunction in the FXS context.

Subject terms: Hippocampus, Molecular neuroscience, Autism spectrum disorders

Introduction

Fragile X syndrome (FXS) is the most common monogenic origin of inherited intellectual disability and autism spectrum disorder (ASD) [1]. It is caused by a trinucleotide CGG expansion in the 5′ UTR of the FMR1 gene, which leads to the loss of expression of the fragile X messenger ribonucleoprotein 1 (FMRP) [2]. People with FXS present heterogeneous symptoms, including, but not limited to, memory impairment, hyperactivity, impulsivity, gastrointestinal dysfunction or autistic-like behavior [3, 4].

The Fmr1 gene knockout (FX) mouse model recapitulates the main traits of the disorder [5]. Notably, an uncontrolled activity of the metabotropic receptor mGluR5, revealed by an enhanced mGluR5-dependent long-term depression (LTD), has been described in FX mice [6, 7]; as well as alterations in dendritic spine density and morphology, with an increase in the immature dendritic spines [8, 9]. In addition to these neuronal traits, the FX mice show gut microbiota dysbiosis [10].

The endocannabinoid system is a key modulator of synaptic plasticity and cognitive function [11], which is affected in FXS. It was previously reported that systemic cannabinoid type-1 receptor (CB1R) inhibition using genetic or pharmacological approaches ameliorated cognitive deficits in FX mice, as well as the aberrant mGluR5-LTD and the altered CA1 spine density in the hippocampus [8, 12]. However, systemic CB1R inhibition was found to induce psychiatric side effects when used as an anti-obesity treatment [13]. Diverse strategies have been developed to avoid these hypothetical central adverse effects of CB1R inhibition, including CB1R peripherally-restricted antagonists and allosteric modulators. We recently demonstrated that AM6545, a peripherally-restricted CB1R antagonist, enhances memory performance in naïve mice [14, 15]. Therefore, we aimed to explore the potential beneficial effects of AM6545 in alleviating key alterations of FX mice.

The present study shows that AM6545 treatment ameliorates object-recognition memory and improves cellular and structural alterations in FX mice. Furthermore, we described a decrease in Akkermansia and Lactobacillus genera in FX mice that increase after AM6545 treatment alongside with an increase in mucine 2 (Muc2) expression in the cecum. Interestingly, we found that the upregulation of central and peripheral serotonin receptor 4 in FX mice after the treatment is needed for sub-chronic AM6545 mnemonic effect. Together, this evidence disclosed the inhibition of peripheral CB1R to alleviate the cognitive deficits in a mouse model for FXS through serotonergic mechanisms.

Materials and methods

Ethics

Animal procedures were conducted following “Animals in Research: Reporting Experiments” (ARRIVE) guidelines and standard ethical guidelines [16, 17] (European Directive 2010/63/EU), and were approved by the local ethical committee (Comitè Ètic d’Experimentació Animal-Parc de Recerca Biomèdica de Barcelona, CEEA-PRBB).

Animals

FX male mice in Friend Virus B background (Fmr1 KO, FVB.129P2-Pde6b+Tyrc-chFmr1tm1Cgr/J) and wild-type mice (WT, FVB.129P2-Pde6b+Tyrc-ch/AntJ) were purchased from The Jackson Laboratory and crossed to obtain outbred FX and WT littermates. FX male mice in C57BL/6J congenic background (B6.129P2-Fmr1tm1Cgr/J) were obtained from the Baylor College of Medicine Mouse Facility and crossed to generate inbred FX and WT littermates. The transgenic line Tg(Thy1-EGFP) MJrs/J (Stock #007788, The Jackson Laboratories, USA) crossed to FX mice in C57BL/6J background, was used to study the density and morphology of dendritic spines. Young-adult male mice (12–14 weeks) used in this study were housed in Plexiglas cages (2–4 mice/cage) and maintained in temperature (21 ± 1 °C) humidity (55 ± 10%) and light (light on at 8 am; light off at 8 pm) controlled environment. Food and water were available ad libitum. Experiments were performed during the light phase after animals were handled for 1 week and habituated to the experimental room. Mice were randomly assigned to the different control and treatment groups. Experiments were conducted by an observer blind to experimental conditions.

Drugs and treatments

AM6545 (1 mg/kg) from Tocris-Bio-Techne was dissolved in 0.26% DMSO, 4.74% ethanol, 5% cremophor-EL and 90% saline. Sotalol (10 mg/kg) from Sigma-Aldrich was dissolved in saline [14]. GR125487 (10 mg/kg) from Tocris-Bio-Techne was dissolved in saline. Drugs were injected intraperitoneally (i.p.) in a volume of 10 ml/kg of body weight for acute or sub-chronic (7d) treatment.

Novel object-recognition test

NORT was performed as previously described [14]. Briefly, on day 1, mice were habituated to a V-shaped maze (V-maze) for 9 min. On day 2 (familiarization phase), two identical objects (familiar objects) were located at the end of each corridor and mice were left to explore them for 9 min. The test phase, also of 9 min, was performed 24 h later, and one of the familiar objects was replaced by a new object (novel object). The time spent exploring each of the objects in the test phase was manually computed to calculate a discrimination index (DI = [Time Novel Object − Time Familiar Object]/[Time Novel Object + Time Familiar Object]). Object exploration time was considered when the orientation of the nose was towards the object. A higher discrimination index is considered to reflect greater memory retention for the familiar object. For sub-chronic treatment, the last AM6545 administration was conducted after the familiarization phase (Supplementary Fig. 1A). GR125487 or sotalol were administered acutely after the familiarization phase and, when needed, 20 min before the last AM6545 administration (Supplementary Fig. 1B-C).

Slice preparation for electrophysiological recordings

Twenty-four hours after the last administration of AM6545 (1 mg/kg, 7d, i.p.), WT and FX mice were anesthetized with isoflurane (5%) and decapitated for slice preparation. Hippocampal slices were prepared as previously described [18–20].

Electrophysiological recordings

Whole-cell patch-clamp recordings were made from pyramidal cells located in the CA1 field of the hippocampus. Cells were patched under visual guidance by infrared differential interference contrast microscopy and verified to be pyramidal neurons by their characteristic voltage response to a current step protocol. Neurons were recorded in voltage-clamp configuration with a patchclamp amplifier (Multiclamp 700B), and data were acquired using pCLAMP 10.2 software (Molecular Devices, California). Patch electrodes were pulled from borosilicate glass tubing and had a resistance of 4–7 MΩ when filled with (in mM) CsCl, 120; HEPES, 10; NaCl, 8; MgCl2, 1; CaCl2, 0.2; EGTA, 2; and QX-314, 20 (290 mOs/ml, pH 7.2–7.3). Cells were excluded from analysis if the series resistance changed by more than 15% during the recording. Recordings were low-pass filtered at 3 kHz and acquired at 10 kHz. mGluR5-LTD was induced by treating slices with 3,5-dihydroxyphenylglycerine (DHPG) (100 μM) for 10 min. LTD was quantified by comparing the mean EPSC amplitude over the 30 min post-drug period with the mean EPSC amplitude during the baseline period and calculating the percentage change from the last 5 min.

Brain slice preparation for dendritic spine analysis

Twenty-four hours after the last administration of AM6545 (1 mg/kg, 7d, i.p.), mice were deeply anesthetized by i.p. injection (0.2 ml/10 g of body weight) of a mixture of ketamine/xylazine (100 mg/kg and 20 mg/kg, respectively) before intracardiac perfusion of cold 4% paraformaldehyde. Brains were removed and stored in a solution of 30% sucrose at 4 °C. Coronal frozen sections were made at 60 μm on a freezing microtome (Leica) and mounted for image acquisition.

Dendritic spine analysis

Secondary and tertiary apical dendrites of pyramidal neurons from the stratum radiatum of CA1 region of the dorsal hippocampus were analyzed. Images were acquired with a confocal microscope (TCS SP5 STED Leica) using a glycerol immersion lens plus 3 times magnification (63X/3) with a sequential line scan at 1024 × 1024 pixel resolution. Serial optical sections were acquired with a 0.13 μm step size. A minimum of 8 dendrites per animal were selected from 4 different slices. A maximum of 2 dendrites were chosen from the same neuron. Confocal images were deconvoluted using Huygens Essential software, and dendritic spines were analyzed and classified using the semiautomated NeuronStudio software with the parameters described (Supplementary Table 1). Spine density was calculated by expressing the average number of spines in a 10 μm portion of the dendrite. Each spine was checked manually by an observer blind to experimental conditions for unbiased classification.

Sample preparation and 16S rRNA gene profiling of fecal microbiota analysis

Fresh fecal samples were collected from each experimental group (Supplementary Table 2) and stored at −80 °C until used. Fecal pellets were diluted 1/10 (w/v) in phosphate buffer saline and vigorously homogenized by vortexing. Cell pellets were isolated by centrifugation at 10,000 rpm for 15 min and used for bacterial DNA isolation using QIAamp DNA stool kit (Qiagen, Germany) following manufacturer’s instructions, including a bead-beating step in a Fastprep (MP Biomedicals, California). The isolated DNA was used as template for partial sequencing (hypervariable V3-V4 region) of the 16S rRNA gene on the Illumina platform following in-house protocols (Novogene Co.) [21]. Data from 16S profiling gut microbiota were total sum scaling and cumulative sum scaling normalization for diversity and abundance analyses, respectively [22]. α-diversity was studied by Chao 1 and Shannon indexes calculated with QIIME.

Tissue preparation for mRNA analysis

Twenty-four hours after the last AM6545 administration (1 mg/kg, 7d, i.p.), mice were euthanized by cervical dislocation to obtain intestinal and hippocampal tissues. The abdomen was cut open for the dissection of ~1 cm of small intestine, cecum and colon. Small intestine was dissected between 1–5 cm distal from pyloric constriction and colon between 4–5 cm distal from cecum. Luminal contents were flushed out with cold phosphate buffer solution 0.1 M from each region with a syringe and a round-tip feeding needle, and dried on paper. Hippocampal tissues were dissected on ice. All tissues were frozen on dry ice and stored at −80 °C until used.

RNA extraction from frozen tissues

Frozen gastrointestinal tissue was minced in lysis buffer from the RNeasy Mini kit (Qiagen) using a homogenizer (Polytron PT2500E) and hippocampal synaptoneurosomes-enriched fractions were obtained as previously described [23]. RNA extraction was performed using the RNeasy Mini kit according to the manufacturer’s instructions, and RNA concentration and integrity were measured using an Agilent 2100 Bioanalyzer (Agilent Technologies) and NanoDrop (Thermo Fisher Scientific).

RNA-seq and differential gene expression analysis

RNA libraries from hippocampal synaptoneurosomes were obtained and processed by Macrogen as previously described [15]. Raw data are available in GEO under the accession number GSE278037.

Differential gene expression (DGE) between experimental groups (Supplementary Table 2) was analyzed using DESeq2 (v1.30) [24] with the cut-off set on |log2fold change| > 0.2 and p-value < 0.1 after Wald test correction procedure. Custom scripts used for data analysis are available from the corresponding author upon reasoned request.

Reverse transcription and quantitative real-time PCR analysis

Reverse transcription and quantitative real-time PCR (qRT-PCR) were performed for small intestine, cecum, colon and hippocampal synaptoneurosomes RNA as previously described [15].

Quantification was performed using the comparative CT Method, relative expression of each gene (Supplementary Table 3) was normalized to Hprt and 18S for gastrointestinal and hippocampal cDNA respectively and the fold change was calculated using the 2(–ΔΔCt) formula [25].

Statistical analysis

Data analyses were performed using GraphPad Prism 10, RStudio (v4.0), MicrobiomeAnalyst and IBM SPSS (v26.0) software. Sample size (n value) is defined by the number of observations (i.e., mice or slices) and is indicated in the legends. Statistical comparisons were evaluated using unpaired Student’s t-test for two groups comparisons, Mann-Whitney U test for two groups comparisons in microbiota experiments (non-normally distributed data), linear mixed-effects models in spine morphology experiments (dependent data) or two-way ANOVA for multiple comparisons. Subsequent Bonferroni or Holm post hoc were used when required (significant interaction between factors). Although no statistical methods were used for power calculation, group sizes were estimated based on previous experiments and literature. Results were expressed as mean ± s.e.m. Comparisons were considered statistically significant when p < 0.05.

Results

AM6545 treatment ameliorates memory deficits in FX mice

First, we tested whether sub-chronic AM6545 treatment could improve memory deficits in FX mice. WT and FX mice received the last administration of a sub-chronic treatment with AM6545 (1 mg/kg, 7d) immediately after the familiarization phase in the NORT. The test phase was performed 24 h later. AM6545 ameliorated cognitive impairment in FX mice both in FVB.129 (two-way ANOVA, interaction: F(1,38) = 4.723, p = 0.036; Bonferroni post hoc, WT-VEH vs. FX-VEH p = 0.005; FX-VEH vs. FX-AM6545 p = 0.001) (Fig. 1A) and C57BL/6J genetic background (two-way ANOVA, interaction: F(1,40) = 10.49, p = 0.002; Bonferroni post hoc, WT-VEH vs. FX-VEH p = 0.0004; FX-VEH vs. FX-AM6545 p = 0.0004) (Fig. 1B), reinforcing the significance of the pro-cognitive effect in both backgrounds.

Fig. 1. Cognitive impairment in FX mice is alleviated after sub-chronic AM6545 treatment.

Fig. 1

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Discrimination index of WT and FX mice after sub-chronic administration of vehicle (VEH) or AM6545 (1 mg/kg, 7d) in A FVB.129 (n = 9–12) and B C57BL/6J (n = 10–13) genetic background. Data are expressed as mean ± s.e.m. ** p < 0.01, *** p < 0.001 (genotype effect); ## p < 0.01, ### p < 0.001 (treatment effect) by two-way ANOVA followed by Bonferroni post hoc.

No differences in total exploration time were observed between pharmacological treatments or genotypes (Supplementary Fig. 2) discarding confounding effects on exploratory behavior. Thus, sub-chronic AM6545 treatment prevents the cognitive deficits in object-recognition memory observed in FX mice from two different genetic backgrounds.

Sub-chronic AM6545 treatment restores cellular alterations in FX hippocampus

The functional and structural synaptic alterations in FX mice that contribute to impairments in synaptic plasticity, include aberrant mGluR5-LTD [6] and alterations in dendritic spines [26]. First, to test whether AM6545 treatment would restore the elevated hippocampal mGluR5-LTD, WT and FX mice were treated with AM6545 (1 mg/kg, 7d) or vehicle. The last AM6545 administration was performed 24 h before brain extraction and hippocampal slice preparation for electrophysiological recording. After 10 min of baseline recording, bath application of DHPG (100 μM, 10 min) induced a transient acute depression, which is considered an electrophysiological readout of group I mGluR activation, followed by a small LTD in WT mice (Fig. 2A). However, we observed that the LTD induced by DHPG was significantly enhanced in vehicle-treated FX mice, which is consistent with previous reports on functional alterations in FXS [27]. Interestingly, we found that sub-chronic AM6545 treatment significantly ameliorated the enhanced mGluR5-LTD in hippocampal slices of FX mice (two-way ANOVA, interaction: F(1,23) = 4.769 p = 0.043; Bonferroni post hoc, WT-VEH vs. FX-VEH p = 0.002; FX-VEH vs. FX-AM6545 p = 0.003) (Fig. 2A-B).

Fig. 2. Sub-chronic AM6545 treatment ameliorates aberrant mGluR5-dependent LTD and normalizes dendritic spines alterations in FX mice.

Fig. 2

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A Average time courses of the change in the amplitude of evoked excitatory postsynaptic currents (eEPSC) in hippocampal slices from FVB.129 mice treated for 7d with vehicle (VEH) or AM6545 (1 mg/kg, 7d) after bath application of DHPG (n = 6–7 mice). B Mean EPSC amplitude values of the last 5 min of recordings in A. C Quantification of the total number of spines in 10 μm of CA1 pyramidal dendrites of C57BL/6J WT and FX mice treated for 7d with VEH or AM6545 (n = 4–5 mice). D Morphological analysis of dendritic spines in the dendrites of pyramidal CA1 neurons in C57BL/6J WT and FX mice after pharmacological treatments (n = 4–5 mice). Each dot represents a hippocampal slice. Data are expressed as mean ± s.e.m. ** p < 0.01 (genotype effect); # p < 0.05 (treatment effect); by linear mixed-effect model followed by Holm post hoc.

Then, taking advantage of FX transgenic mice expressing the EGFP fluorescent protein under neuron-specific Thy1 promoter (Thy1-EGFP mice), we analyzed dendritic spine density and morphology of apical dendrites from CA1 24 h after the last administration of sub-chronic AM6545 treatment. Vehicle-treated FX mice showed an increase in dendritic spine density compared to their WT littermates, that was normalized after sub-chronic AM6545 treatment (linear mixed-effect model, interaction: F(3,11.33) = 11.333 p = 0.0035; Holm post hoc, WT-VEH vs. FX-VEH p = 0.008; FX-VEH vs. FX-AM6545 p = 0.031) (Fig. 2C). Finally, we further classified the spines based on their morphology. We found that vehicle-treated FX mice presented an elevated density of thin (immature) spines that showed a non-significant trend to be restored after sub-chronic AM6545 treatment (linear mixed-effect model, interaction: F(3,13.69) = 7.078 p = 0.0041; Holm post hoc, WT-VEH vs. FX-VEH p = 0.0078; FX-VEH vs. FX-AM6545 p = 0.05) (Fig. 2D). Therefore, sub-chronic AM6545 treatment normalizes the aberrant mGluR5-dependent LTD and dendritic spine density of CA1 pyramidal neurons in FX mice.

Fecal microbiome and intestinal Muc2 expression of FX mice is sensitive to AM6545 treatment

To explore whether AM6545 could modify the gut microbiome, we investigated fecal microbiota in FX mice, and whether sub-chronic AM6545 treatment would modify fecal microbiota composition. To this end, we performed 16S rRNA gene amplicon sequencing of feces samples in the same cohort of mice before and after vehicle or AM6545 administration, and analyzed the changes related either to the genotype or the treatment by paired comparisons (Supplementary Table 2). In terms of structural differences, no major changes were observed in α-diversity between the groups analyzed (Supplementary Fig. 3).

We observed differences in fecal microbiome composition between WT and FX mice in basal conditions, such as the reduction in Lactobacillus genus (Fig. 3A, Supplementary Table 4). Then, after behavioral experiments and sub-chronic vehicle administration, we found more changes between genotypes than in basal conditions. These changes included the reduction in the relative abundance of Akkermansia genus in vehicle-treated FX mice (Fig. 3B, Supplementary Table 5) compared to vehicle-treated WT mice. Interestingly, sub-chronic AM6545 treatment results in an increase in bacteria belonging to both Akkermansia and Lactobacillus genera (Fig. 3C, Supplementary Table 6); pointing to the ability of AM6545 treatment to restore microbiota alterations observed in FX mice.

Fig. 3. Fecal microbiome and Muc2 expression changes in FX mice after sub-chronic AM6545 treatment.

Fig. 3

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A-B Volcano plot showing differential abundance of microbiome composition in A C57BL/6J FX non-manipulated mice compared to WT non-manipulated mice (WT-basal vs. FX-basal), B C57BL/6J vehicle-treated FX mice compared to vehicle-treated WT mice (WT-VEH vs. FX-VEH) and C C57BL/6J FX mice sub-chronically treated with AM6545 (1 mg/kg, 7d) compared to vehicle-treated FX mice (FX-VEH vs. FX-AM6545). Orange and green dots indicate enriched or depleted species in each comparison in terms of relative abundance at genus level. (WT-basal, n = 19; FX-basal, n = 20; WT-VEH, n = 10; FX-VEH, n = 10; FX-AM6545, n = 10). Significance is set at p < 0.05. D-F Gastrointestinal mRNA levels of Htr4 in D small intestine, E cecum and F colon of C57BL/6J FX mice treated for 7d with vehicle (VEH) or AM6545 (1 mg/kg, 7d) (n = 4–5). Data are expressed as mean ± s.e.m. # p < 0.05 (treatment effect) by one-way ANOVA followed by Bonferroni post hoc.

In addition, given the alterations in gastrointestinal dysfunction described in FXS [4] we next determine whether the epithelial gastrointestinal integrity was compromised by quantifying Muc2 expression in the small intestine, cecum and colon of WT- and FX-vehicle treated mice and AM6545-treated FX mice (Fig. 3D-F). We found that Muc2 mRNA expression level was increased in the cecum of FX mice after sub-chronic AM6545 administration (one-way ANOVA, interaction: F(2,11) = 4.843 p = 0.031; Bonferroni post hoc, FX-VEH vs. FX-AM6545 p = 0.039) (Fig. 3E). This suggests, that AM6545 may promote Muc2 expression.

Serotonin receptor 4 gene is upregulated in FX mice after sub-chronic AM6545 treatment

To explore more in depth the synaptic impact of AM6545 in the context of FXS, we performed mRNA transcriptome analysis of hippocampal synaptoneurosomes. We treated FX mice for 7d with AM6545 or vehicle and obtained hippocampal samples 24 h after the last AM6545 administration, just after the test phase of the NORT. DGE analysis revealed 7 genes significantly upregulated in FX mice treated with AM6545, whereas only Dcp2, was downregulated (Fig. 4A, Supplementary Table 7).

Fig. 4. Sub-chronic AM6545 treatment upregulates serotonin 4 receptor in the hippocampus and cecum of FX mice.

Fig. 4

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A Volcano plot showing differentially expressed genes in C57BL/6J FX mice treated with vehicle (VEH) or AM6545 (1 mg/kg, 7d). Red and blue dots represent significantly up or downregulated genes, respectively. (FX-VEH, n = 5; FX-AM6545, n = 4). Cut-off was set at |log2FC| >0.2 and adjusted p-value < 0.1. mRNA levels of Htr4 in B hippocampal synaptoneurosomes, C small intestine, D cecum and E colon of C57BL/6J FX mice treated for 7d with vehicle (VEH) or AM6545 (1 mg/kg, 7d) (n = 4–10). Data are expressed as mean ± s.e.m. # p < 0.05 (treatment effect) by Student’s t-test.

Among the upregulated genes, we focused on Htr4, since Htr4/5-HT4R expression/function is associated with hippocampal plasticity and processing of information [28]. We used an independent cohort of FX mice sub-chronically treated with AM6545 or vehicle, to validate the RNA seq result. We found, using qRT-PCR, that Htr4 was upregulated in hippocampal synaptoneurosomes from AM6545-treated FX mice (Student’s t-test: p = 0.048) (Fig. 4B).

Since Htr4 expression is localized to the brain and gastrointestinal tissues [29], and considering the peripherally restricted profile of AM6545, we investigated the effects of AM6545 sub-chronic administration on the gastrointestinal tract of FX mice. We found a significant upregulation of Htr4 mRNA levels in the cecum of AM6545-treated FX mice (Student’s t-test: p = 0.039) (Fig. 4D) whereas no changes were detected in the small intestine or colon (Fig. 4C, E). Thus, sub-chronic AM6545 administration specifically upregulates cecal Htr4 levels without affecting other intestinal segments.

Interestingly, we found a positive correlation between discrimination index and Htr4 levels from both RNA-sequencing (Pearson’s correlation coefficient = 0.858, p = 0.003) (Supplementary Fig. 4A) and qRT-PCR data (Pearson’s correlation coefficient = 0.485, p = 0.048) (Supplementary Fig. 4B). This indicates that serotonin receptor 4 mRNA levels increase in FX mice after AM6545 administration in a way that relates to the performance in NORT.

Cognitive rescue of sub-chronic AM6545 in FX mice is dependent of a serotonergic mechanism

Based on Htr4 correlation with memory in hippocampus, we hypothesized a possible implication of 5-HT4R in the alleviation of the cognitive impairment in FXS. To assess whether sub-chronic AM6545 treatment improves memory with the participation of a serotonergic signaling mechanism, FX mice were treated during 7d with vehicle or AM6545 and on the last day of treatment, they were pre-treated with the 5-HT4R antagonist GR125487 (10 mg/kg). Thus, immediately after the familiarization phase in the NORT, FX mice received an acute administration of GR125487 or saline, 20 min before the last AM6545 administration. When the test phase was performed 24 h later, we observed that the pre-treatment with GR125487 abolished the mnemonic effect of sub-chronic AM6545 treatment in FX mice (two-way ANOVA, interaction: F(1,34) = 6.221, p = 0.018; Bonferroni post hoc, FX-saline-VEH vs. FX-saline-AM6545 p = 0.013; FX-saline-AM6545 vs. FX-GR125487-AM6545 p = 0.037) (Fig. 5A). Then, since we previously demonstrated that acute AM6545 improves memory persistence through a peripheral adrenergic mechanism in naïve mice [14], we explored whether such a mechanism could also be involved in the mnemonic effects of sub-chronic AM6545 in FX mice. Notably, we found that the sub-chronic alleviation of memory deficits after AM6545 treatment was not abolished by acute pre-treatment with the peripherally-restricted β-adrenergic antagonist sotalol (10 mg/kg) when administered 20 min before the last AM6545 administration in FX mice (Fig. 5B).

Fig. 5. A serotonergic mechanism mediates the cognitive improvement of FX mice after sub-chronic AM6545 treatment.

Fig. 5

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A Discrimination index of C57BL/6J FX mice after sub-chronic vehicle (VEH) or AM6545 (1 mg/kg, 7d) administration and pre-treatment with saline or GR125487 (10 mg/kg) after the familiarization phase of the NORT (n = 8–10). B Discrimination index of C57BL/6J FX mice after sub-chronic AM6545 (1 mg/kg, 7d) administration and pre-treatment with saline or sotalol (10 mg/kg) after the familiarization phase of the NORT (n = 4–5). Data are expressed as mean ± s.e.m. + p < 0.05 (GR125487 pre-treatment effect); # p < 0.05 (treatment effect) by two-way ANOVA followed by Bonferroni post hoc.

Conversely, the acute mnemonic effect of AM6545 was not prevented by the administration of GR125487 (Supplementary Fig. 6A) in FX mice after the familiarization phase (Supplementary Fig. 6B) but was significantly inhibited by acute pre-treatment with sotalol (Supplementary Fig. 6D) in FX mice (Student’s t-test: p < 0.000001) (Supplementary Fig. 6E). These effects were not related to modifications in the exploratory behavior since total exploration time did not significantly change between treatments (Supplementary Fig. 5, Supplementary Fig. 6C), although there was a slight modification after sotalol pre-treatment before acute AM6545 administration (Supplementary Fig. 6F) that did not affect discrimination index.

Together this indicates a mechanistic divergence based on treatment duration. While the acute effect of AM6545 is mainly driven by peripheral noradrenergic signaling, the effect that sub-chronic AM6545 has on FX mice memory performance involves 5-HT4R modulation.

Discussion

In the present study we described that AM6545, a CB1R antagonist largely restricted to the periphery, rescues memory impairment, synaptic abnormalities and gut microbiota composition in FX mice. Moreover, we decipher that the memory rescue depends on peripheral β-adrenergic and 5-HT4R signaling in this FXS mouse model.

Previous research in our group has pointed to the endocannabinoid system as a potential therapeutic target in the treatment of FXS-associated cognitive deficits. Specifically, low doses of the systemic CB1R antagonist rimonabant, and the neutral CB1R antagonist NESS0327 showed improvements in several traits of FX mice such as the memory impairment, the elevated mGluR5-LTD, the alterations in dendritic spines or the susceptibility to audiogenic epileptic seizures [8, 12]. Here, we aimed to target peripheral CB1R in the FXS mouse model, as we previously reported that the memory-enhancing effects of systemic CB1R inhibition with rimonabant in naïve mice can be reproduced by peripheral CB1R antagonists such as AM6545 [14, 15].

First, we assessed memory performance in the NORT in mice that were sub-chronically treated with AM6545. Under these conditions, we found a significant amelioration of the memory deficits after the 7d treatment in two different genetic backgrounds, one outbred (FVB.129) and another inbred (C57BL/6J). This finding indicates a genuine overall effect of AM6545 that is not associated to a specific genetic background, similar to previously described in inbred and outbred strains of naïve mice [15].

Interestingly, AM6545 normalizes the enhanced hippocampal mGluR5-LTD of FX mice and also restores the increased density of immature spines in the hippocampus. These cellular alterations have been well-described in FX mice [6, 26] and have been typically associated to cognitive impairment in FXS [30]. The changes in synaptic function and structure of AM6545 are somehow reminiscent to the effect of sub-chronic treatment with rimonabant in which mGluR5-LTD and the number of immature spines in the hippocampus of FX mice were normalized [8, 12]. Overall, these effects on synaptic plasticity in FX mice are consistent with the unanticipated hypothesis that rimonabant systemic effects can also be obtained targeting peripheral CB1R.

Gastrointestinal disorders are frequently reported in ASD and FXS, including symptoms such as increased gut permeability, dysbiosis and inflammation [4]. These alterations may contribute to behavioral and cognitive impairments via the gut–brain axis and targeting the gut microbiome may be a potential strategy to improve gastrointestinal and behavioral symptoms in individuals with ASD [31–33]. Given that AM6545 is a largely peripheral antagonist [34] and the emerging role of the endocannabinoid system in modulating the gut-brain axis [35], we next analyzed fecal microbiome composition. Here we reported a significant decrease in Lactobacillus and Akkermansia population in feces from FX mice compared to WT littermates. Notably, we also found that both reductions in abundance were restored after AM6545 treatment. Both genera have been recently pinpointed by others in a similar context. On the one hand, treatment with Lactobacillus in mouse models of ASD restored social cognitive deficits and synaptic functions [36, 37]. Indeed, a clinical trial is ongoing to evaluate the beneficial effects of increasing Lactobacillus species in children with FXS (NCT06279858). On the other hand, there is a dual role of Akkermansia in neurodevelopmental and neurodegenerative disorders [35]. In conditions like ASD and FXS, elevated levels of mucin-degrading bacteria, like Verrucomicrobiota and Akkermansia muciniphila, can act as biomarkers for gut barrier integrity, permeability and inflammation [10, 38]. While A. muciniphila may help restore gut function and reduce permeability, it has also inflammatory potential when the mucosal layer is compromised. Nevertheless, treatment with A. muciniphila in a nonalcoholic steatohepatitis rat model reversed cognitive impairment [39]. Indeed, fecal microbiota transplantation from WT mice into a FX mouse model increased A. muciniphila levels, while ameliorating cognitive and social deficits [40, 41]. This makes it unclear if A. muciniphila is beneficial or detrimental in the context of FXS. Although further research is needed to elucidate the role of these genera in FXS, some of the effects of AM6545 administration could be attained through the microbiota-gut-brain axis [42].

Additionally, we found that the increase in Akkermansia and Lactobacillus in AM6545-treated FX mice is accompanied by the increase of Muc2 expression in the cecum. Mucin 2 is the primary mucin secreted from globet cells and is essential for maintaining the gut integrity and inflammation [43]. Indeed, there is a link between the restoration of gastrointestinal barrier integrity and cognitive performance [44]. Thus, by promoting Muc2 expression, AM6545 likely stabilizes the gut brain barrier, allowing Akkermansia to contribute to a homeostatic rather than inflammatory environment. Therefore, by modulating the microbiota-gut-brain axis including microbiota composition and cecal integrity by Muc2 expression, AM6545 could offer a promising multi-level approach to alleviate symptoms in FXS.

Our transcriptomic analysis of hippocampal synaptoneurosomes revealed few changes in gene expression. Dcp2 downregulation, could indicate a higher concentration of this mRNA decapping protein in P-bodies [45], facilitating its interaction with excessive mRNAs found in FX synapses. Interestingly, we found the upregulation of hippocampal Htr4 in FX mice that received AM6545, strongly indicating a possible role of 5-HT4R in AM6545-mediated hippocampal changes. Importantly, 5-HT4R plays a relevant role in hippocampus-dependent learning and memory, and the activation of these receptors has been related with cognitive enhancement [28]. The administration of 5-HT4R agonists enhance memory in naïve mice [46, 47] and ameliorate cognitive deficits in mouse models of Alzheimer´s disease [48, 49], while 5-HT4R decreased density has been associated to cognitive impairments [50, 51].

Interestingly, the hippocampal upregulation of Htr4 is accompanied by the upregulation of Htr4 in the gastrointestinal tract, specifically in the cecum; suggesting a possible synergy between the gut and the brain by which AM6545 is effective in FXS. While Htr4 is well-known for enhancing cognition and structural plasticity in the central nervous system [52], its upregulation in the cecum is not well-understood. Previous research has established a functional link between increased Muc2 levels and Htr4 expression [53], suggesting that serotonergic signaling may also play a role in maintaining mucosal homeostasis. Notably, Lactobacillus species, which were increased in our AM6545-treated FX mice, have been shown to promote the gastrointestinal expression of Htr4 and enhance mucin 2 production [54]. These changes suggest that the pro-cognitive effects of AM6545 may be driven by a periphery-to-central serotonin signaling cascade that simultaneously modifies signs of the gut barrier integrity and enhances hippocampal function in FX mice.

Moreover, dysregulation in serotonin modulation has also been reported in ASD and FXS [55, 56]. Recent studies reported a reduced expression of 5-HT1BR in subjects with FXS [57] and lower binding activity of 5HT1AR in FX mice [58]. Interestingly, the modulation of specific serotonin receptors (5-HT1R, 5-HT2R, 5-HT5R and 5-HT7R) has been described to ameliorate behavior and synaptic plasticity in FXS models [59–63]. However, changes in 5-HT4R have not been previously associated with FXS.

Finally, to further understand the role of 5-HT4R in AM6545 effect in FXS, we addressed whether the activation of 5-HT4R may interfere with memory performance in FX mice. Acute pre-treatment with the 5-HT4R antagonist GR125487, abolished the pro-cognitive effect of the sub-chronic AM6545 treatment in FX mice. Then, since we previously demonstrated that AM6545 improves memory persistence through a peripheral adrenergic mechanism in naïve mice [14, 15], we explored whether such a mechanism could also be involved in FX mice. However, we found that acute pre-treatment with the peripherally-restricted β-adrenergic antagonist sotalol, did not abolish the pro-cognitive effect of the sub-chronic AM6545 treatment in FX mice. In contrast, the acute mnemonic effect of AM6545 appeared to rely on a noradrenergic mechanism since pre-treatment with sotalol, but not GR125487, blocked the cognitive alleviation in FX mice.

These experimental approaches indicate a shift in the pharmacological recruitment of AM6545 in FX mice, transitioning from an initial peripheral adrenergic response to a 5-HT4R-dependent pathway upon sub-chronic administration. In this regard, previous studies demonstrated a significant interaction between serotonergic and endocannabinoid systems [64]. Genetic or pharmacological CB1R inhibition has been shown to enhance serotonin levels and control the function and expression of serotonin receptors [65–67]. Specifically, low doses of 5-HT4R agonists reverse CB1R-induced memory impairment [68]. Moreover, activation of 5-HT4Rs has been described to increase intracellular cAMP levels, induce LTP and regulate neurotransmitter release in hippocampus [69, 70]; which may explain why its activation is needed for the mnemonic effect of AM6545 in FX mice.

Altogether our results suggest that the pharmacological inhibition of CB1R with the largely peripheral compound AM6545, alleviates significant central and peripheral alterations in the FX mouse and points to the 5-HT4R as a potential central effector in the amelioration of cognitive dysfunction. Furthermore, the modification of these serotonergic receptors important for cognitive performance in the context of FXS, opens the possibility to explore peripheral treatments to ameliorate intellectual disability disorders.

Supplementary information

Supplementary Information (4MB, docx)

Acknowledgements

We are grateful to Raquel Martín, Marta Linares and Dulce Real for expert technical assistance, the Genomics Core Facility (MELIS-UPF) and the Scientific Computing Core Facility (MELIS-UPF) for their infrastructure and assistance and Neurobiology of Behaviour Research Group (Grenec-NeuroBio) for their experimental assistance.

Author contributions

LR-R, Behavioral and biochemical experiments, transcriptomic study, statistical analysis, graphs and writing of the manuscript; AB-M, Behavioral and biochemical experiments, statistical analysis and writing of the manuscript; SM-T, Behavioral experiments. IM-G, PL-R, AG-M and AR-M, Electrophysiological experiments; SA and MG, Microbiota experiments RM, Supervision and funding of the study AO, Conceptualization, supervision, funding of the study and writing of the manuscript. All authors revised the final version of the manuscript.

Funding

LR-R was supported by a predoctoral fellowship from Spanish Ministry of Science and Innovation (PRE2019-087644). AB-M was supported by a predoctoral fellowship from Spanish Ministry of Universities (FPU20/02061). IM-G was supported by a predoctoral fellowship from Spanish Ministry of Science, Innovation and Universities (FPU17/04283). This work was supported by the Spanish Ministry of Science and Innovation (PID2021-123482OB-I00), La Marató de TV3 Foundation (202224-30-31) and FRAXA Research Foundation (04-3222167) to AO; the Spanish Agencia Estatal de Investigación (PID2022-136597NB-I00) and the Junta de Andalucía (P20-0881) to AR-M; the MICIU/AEI/10.13039/501100011033 and European Union NextGenerationEU/PRTR (Grant CNS2023-145550) to SA and MG and Generalitat de Catalunya, AGAUR (2021 SGR 00912) to RM.

Data availability

RNA-seq raw data was deposited into the Gene Expression Omnibus database under accession number GSE278037. Any additional information is available from the corresponding author upon reasoned request.

Competing interests

RM and AO declare intellectual property of the patent PCT/EP2013/055728. The remaining authors declare no conflict of interest.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Lucía de los Reyes-Ramírez, Araceli Bergadà-Martínez.

Supplementary information

The online version contains supplementary material available at https://doi.org/10.1038/s41398-026-04360-4.

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Data Availability Statement

RNA-seq raw data was deposited into the Gene Expression Omnibus database under accession number GSE278037. Any additional information is available from the corresponding author upon reasoned request.

Articles from Translational Psychiatry are provided here courtesy of Nature Publishing Group

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