Mouse Model Of Fragile X Syndrome Research Articles

P2X7 expression patterns in the developing Fmr1-knockout mouse hippocampus.

Fragile-X Syndrome (FXS)is the leading monogenetic cause of intellectual disability among children but remains without a cure. Using the Fmr1 KO mouse model of FXS, much work has been done to understand FXS hippocampus dysfunction. Purinergic signaling, where ATP and its metabolites are used as signaling molecules, participates in hippocampus development, but it is unknown if purinergic signaling is affected in the developing Fmr1 KO hippocampus. In our study, we characterized the purinergic receptor P2X7. We first found that P2X7 was reduced in Fmr1 KO whole hippocampus tissue at P14 and P21, corresponding to the periods of neurite outgrowth and synaptic refinement in the hippocampus. We then evaluated the cell-specific expression of P2X7 with immunofluorescenceand found differences between WT and Fmr1 KO mice in P2X7 colocalization with hippocampal microglia and neurons. P2X7 colocalized more with microglia at P14 and P21, but there was a sex-specific reduction in P2X7 colocalization with neurons. In contrast, male mice at P14 and P21 showed reduced neuronal P2X7 colocalization compared to females, but only females showed reduced absolute neuronal P2X7 expression across the dorsal hippocampal formation. Together, our results suggest that P2X7 expression is altered during Fmr1-KO hippocampal development, potentially influencing several developmental processes in the Fmr1-KO hippocampus formation.

Dysregulated Purinergic Signalling in Fragile X Syndrome Cortical Astrocytes.

The symptoms of fragile X syndrome (FXS), caused by a single gene mutation to Fmr1, have been increasingly linked to disordered astrocyte signalling within the cerebral cortex. We have recently demonstrated that the purinergic signalling pathway, which utilizes nucleoside triphosphates and their metabolites to facilitate bidirectional glial and glial-neuronal interactions, is upregulated in cortical astrocytes derived from the Fmr1 knockout (KO) mouse model of FXS. Heightened Fmr1 KO P2Y purinergic receptor levels were correlated with prolonged intracellular calcium release, elevated synaptogenic protein secretion, and hyperactivity of developing circuits. However, due to the relative lack of sensitive and reproducible quantification methods available for measuring purines and pyrimidines, determining the abundance of these factors in Fmr1 KO astrocytes was limited. We therefore developed a hydrophilic interaction liquid chromatography protocol coupled with mass spectrometry to compare the abundance of intracellular and extracellular purinergic molecules between wildtype and Fmr1 KO mouse astrocytes. Significant differences in the concentrations of UDP, ATP, AMP, and adenosine intracellular stores were found within Fmr1 KO astrocytes relative to WT. The extracellular level of adenosine was also significantly elevated in Fmr1 KO astrocyte-conditioned media in comparison to media collected from WT astrocytes. Glycosylation of the astrocyte membrane-bound CD39 ectonucleotidase, which facilitates ligand breakdown following synaptic release, was also elevated in Fmr1 KO astrocyte cultures. Together, these differences demonstrated further dysregulation of the purinergic signalling system within Fmr1 KO cortical astrocytes, potentially leading to significant alterations in FXS purinergic receptor activation and cellular pathology.

Therapeutic efficacy of the BKCa channel opener chlorzoxazone in a mouse model of Fragile X syndrome.

Fragile X syndrome (FXS) is an X-linked neurodevelopmental disorder characterized by several behavioral abnormalities, including hyperactivity, anxiety, sensory hyper-responsiveness, and autistic-like symptoms such as social deficits. Despite considerable efforts, effective pharmacological treatments are still lacking, prompting the need for exploring the therapeutic value of existing drugs beyond their original approved use. One such repurposed drug is chlorzoxazone which is classified as a large-conductance calcium-dependent potassium (BKCa) channel opener. Reduced BKCa channel functionality has been reported in FXS patients, suggesting that molecules activating these channels could serve as promising treatments for this syndrome. Here, we sought to characterize the therapeutic potential of chlorzoxazone using the Fmr1-KO mouse model of FXS which recapitulates the main phenotypes of FXS, including BKCa channel alterations. Chlorzoxazone, administered either acutely or chronically, rescued hyperactivity and acoustic hyper-responsiveness as well as impaired social interactions exhibited by Fmr1-KO mice. Chlorzoxazone was more efficacious in alleviating these phenotypes than gaboxadol and metformin, two repurposed treatments for FXS that do not target BKCa channels. Systemic administration of chlorzoxazone modulated the neuronal activity-dependent gene c-fos in selected brain areas of Fmr1-KO mice, corrected aberrant hippocampal dendritic spines, and was able to rescue impaired BKCa currents recorded from hippocampal and cortical neurons of these mutants. Collectively, these findings provide further preclinical support for BKCa channels as a valuable therapeutic target for treating FXS and encourage the repurposing of chlorzoxazone for clinical applications in FXS and other related neurodevelopmental diseases.

Sex-specific loss of mitochondrial membrane integrity in the auditory brainstem of a mouse model of Fragile X syndrome.

Sound sensitivity is one of the most common sensory complaints for people with autism spectrum disorders (ASDs). How and why sounds are perceived as overwhelming by affected people is unknown. To process sound information properly, the brain requires high activity and fast processing, as seen in areas like the medial nucleus of the trapezoid body (MNTB) of the auditory brainstem. Recent work has shown dysfunction in mitochondria, which are the primary source of energy in cells, in a genetic model of ASD, Fragile X syndrome (FXS). Whether mitochondrial functions are also altered in sound-processing neurons, has not been characterized yet. To address this question, we imaged the MNTB in a mouse model of FXS. We stained MNTB brain slices from wild-type and FXS mice with two mitochondrial markers, TOMM20 and PMPCB, located on the Outer Mitochondrial Membrane and in the matrix, respectively. These markers allow exploration of mitochondrial subcompartments. Our integrated imaging pipeline reveals significant sex-specific differences between genotypes. Colocalization analyses between TOMM20 and PMPCB reveal that the integrity of mitochondrial subcompartments is most disrupted in female FXS mice compared to female wildtype mice. We highlight a quantitative fluorescence microscopy pipeline to monitor mitochondrial functions in the MNTB from control or FXS mice and provide four complementary readouts. Our approach paves the way to understanding how cellular mechanisms important to sound encoding are altered in ASDs.

Cage effects on synaptic plasticity and its modulation in a mouse model of fragile X syndrome.

Fragile X syndrome (FXS) is characterized by impairments in executive function including different types of learning and memory. Long-term potentiation (LTP), thought to underlie the formation of memories, has been studied in the Fmr1 mouse model of FXS. However, there have been many discrepancies in the literature with inconsistent use of littermate and non-littermate Fmr1 knockout (KO) and wild-type (WT) control mice. Here, the influence of the breeding strategy (cage effect) on short-term potentiation (STP), LTP, contextual fear conditioning (CFC), expression of N-methyl-d-aspartate receptor (NMDAR) subunits and the modulation of NMDARs, were examined. The largest deficits in STP, LTP and CFC were found in KO mice compared with non-littermate WT. However, the expression of NMDAR subunits was unchanged in this comparison. Rather, NMDAR subunit (GluN1, 2A, 2B) expression was sensitive to the cage effect, with decreased expression in both WT and KO littermates compared with non-littermates. Interestingly, an NMDAR-positive allosteric modulator, UBP714, was only effective in potentiating the induction of LTP in non-littermate KO mice and not the littermate KO mice. These results suggest that commonly studied phenotypes in Fmr1 KOs are sensitive to the cage effect and therefore the breeding strategy may contribute to discrepancies in the literature.This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

Astroglial Kir4.1 potassium channel deficit drives neuronal hyperexcitability and behavioral defects in Fragile X syndrome mouse model

Fragile X syndrome (FXS) is an inherited form of intellectual disability caused by the loss of the mRNA-binding fragile X mental retardation protein (FMRP). FXS is characterized by neuronal hyperexcitability and behavioral defects, however the mechanisms underlying these critical dysfunctions remain unclear. Here, using male Fmr1 knockout mouse model of FXS, we identify abnormal extracellular potassium homeostasis, along with impaired potassium channel Kir4.1 expression and function in astrocytes. Further, we reveal that Kir4.1 mRNA is a binding target of FMRP. Finally, we show that the deficit in astroglial Kir4.1 underlies neuronal hyperexcitability and several behavioral defects in Fmr1 knockout mice. Viral delivery of Kir4.1 channels specifically to hippocampal astrocytes from Fmr1 knockout mice indeed rescues normal astrocyte potassium uptake, neuronal excitability, and cognitive and social performance. Our findings uncover an important role for astrocyte dysfunction in the pathophysiology of FXS, and identify Kir4.1 channel as a potential therapeutic target for FXS.

Phenotypic analysis of multielectrode array EEG biomarkers in developing and adult male Fmr1 KO mice

Fragile X Syndrome (FXS) is a leading known genetic cause of intellectual disability with symptoms that include increased anxiety and social and sensory processing deficits. Recent electroencephalographic (EEG) studies in humans with FXS have identified neural oscillation deficits that include increased resting state gamma power, increased amplitude of auditory evoked potentials, and reduced phase locking of sound-evoked gamma oscillations. Similar EEG phenotypes are present in mouse models of FXS, but very little is known about the development of such abnormal responses. In the current study, we employed a 30-channel mouse multielectrode array (MEA) system to record and analyze resting and stimulus-evoked EEG signals in male P21 and P91 WT and Fmr1 KO mice. This led to several novel findings. First, P91, but not P21, Fmr1 KO mice have significantly increased resting EEG power in the low- and high-gamma frequency bands. Second, both P21 and P91 Fmr1 KO mice have markedly attenuated inter-trial phase coherence (ITPC) to spectrotemporally dynamic auditory stimuli as well as to 40 Hz and 80 Hz auditory steady-state response (ASSR) stimuli. This suggests abnormal temporal processing from early development that may lead to abnormal speech and language function in FXS. Third, we found hemispheric asymmetry of fast temporal processing in the mouse auditory cortex in WT but not Fmr1 KO mice. Together, these findings define a set of EEG phenotypes in young and adult mice that can serve as translational targets for genetic and pharmacological manipulation in phenotypic rescue studies.

MGluR7 allosteric modulator AMN082 corrects protein synthesis and pathological phenotypes in FXS

Fragile X syndrome (FXS) is the leading cause of inherited autism and intellectual disabilities. Aberrant protein synthesis due to the loss of fragile X messenger ribonucleoprotein (FMRP) is the major defect in FXS, leading to a plethora of cellular and behavioral abnormalities. However, no treatments are available to date. In this study, we found that activation of metabotropic glutamate receptor 7 (mGluR7) using a positive allosteric modulator named AMN082 represses protein synthesis through ERK1/2 and eIF4E signaling in an FMRP-independent manner. We further demonstrated that treatment of AMN082 leads to a reduction in neuronal excitability, which in turn ameliorates audiogenic seizure susceptibility in Fmr1 KO mice, the FXS mouse model. When evaluating the animals’ behavior, we showed that treatment of AMN082 reduces repetitive behavior and improves learning and memory in Fmr1 KO mice. This study uncovers novel functions of mGluR7 and AMN082 and suggests the activation of mGluR7 as a potential therapeutic approach for treating FXS.

On-Tissue Spatial Proteomics Integrating MALDI-MS Imaging with Shotgun Proteomics Reveals Soy Consumption-Induced Protein Changes in a Fragile X Syndrome Mouse Model.

Fragile X syndrome (FXS), the leading cause of inherited intellectual disability and autism, is caused by the transcriptional silencing of the FMR1 gene, which encodes the fragile X messenger ribonucleoprotein (FMRP). FMRP interacts with numerous brain mRNAs that are involved in synaptic plasticity and implicated in autism spectrum disorders. Our published studies indicate that single-source, soy-based diets are associated with increased seizures and autism. Thus, there is an acute need for an unbiased protein marker identification in FXS in response to soy consumption. Herein, we present a spatial proteomics approach integrating mass spectrometry imaging with label-free proteomics in the FXS mouse model to map the spatial distribution and quantify levels of proteins in the hippocampus and hypothalamus brain regions. In total, 1250 unique peptides were spatially resolved, demonstrating the diverse array of peptidomes present in the tissue slices and the broad coverage of the strategy. A group of proteins that are known to be involved in glycolysis, synaptic transmission, and coexpression network analysis suggest a significant association between soy proteins and metabolic and synaptic processes in the Fmr1KO brain. Ultimately, this spatial proteomics work represents a crucial step toward identifying potential candidate protein markers and novel therapeutic targets for FXS.

Hyperexcitability in the Olfactory Bulb and Impaired Fine Odor Discrimination in the Fmr1 KO Mouse Model of Fragile X Syndrome.

Fragile X syndrome (FXS) is the single most common monogenetic cause of autism spectrum disorders (ASDs) in humans. FXS is caused by loss of expression of the fragile X mental retardation protein (FMRP), an mRNA-binding protein encoded on the X chromosome involved in suppressing protein translation. Sensory processing deficits have been a major focus of studies of FXS in both humans and rodent models of FXS, but olfactory deficits remain poorly understood. Here, we conducted experiments in wild-type (WT) and Fmr1 knock-out (KO; Fmr1-/y ) mice (males) that lack expression of the gene encoding FMRP to assess olfactory circuit and behavioral abnormalities. In patch-clamp recordings conducted in slices of the olfactory bulb, output mitral cells (MCs) in Fmr1 KO mice displayed greatly enhanced excitation under baseline conditions, as evidenced by a much higher rate of occurrence of spontaneous network-level events known as long-lasting depolarizations (LLDs). The higher probability of spontaneous LLDs (sLLDs), which appeared to be because of a decrease in GABAergic synaptic inhibition in glomeruli leading to more feedforward excitation, caused a reduction in the reliability of stimulation-evoked responses in MCs. In addition, in a go/no-go operant discrimination paradigm, we found that Fmr1 KO mice displayed impaired discrimination of odors in difficult tasks that involved odor mixtures but not altered discrimination of monomolecular odors. We suggest that the Fmr1 KO-induced reduction in MC response reliability is one plausible mechanism for the impaired fine odor discrimination.SIGNIFICANCE STATEMENT Fragile X syndrome (FXS) in humans is associated with a range of debilitating deficits including aberrant sensory processing. One sensory system that has received comparatively little attention in studies in animal models of FXS is olfaction. Here, we report the first comprehensive physiological analysis of circuit defects in the olfactory bulb in the commonly-used Fmr1 knock-out (KO) mouse model of FXS. Our studies indicate that Fmr1 KO alters the local excitation/inhibition balance in the bulb, similar to what Fmr1 KO does in other brain circuits, but through a novel mechanism that involves enhanced feedforward excitation. Furthermore, Fmr1 KO mice display behavioral impairments in fine odor discrimination, an effect that may be explained by changes in neural response reliability.

Cochlear Nucleus Transcriptome of a Fragile X Mouse Model Reveals Candidate Genes for Hyperacusis.

Fragile X Syndrome (FXS) is a hereditary form of autism spectrum disorder. It is caused by a trinucleotide repeat expansion in the Fmr1 gene, leading to a loss of Fragile X Protein (FMRP) expression. The loss of FMRP causes auditory hypersensitivity: FXS patients display hyperacusis and the Fmr1- knock-out (KO) mouse model for FXS exhibits auditory seizures. FMRP is strongly expressed in the cochlear nucleus and other auditory brainstem nuclei. We hypothesize that the Fmr1-KO mouse has altered gene expression in the cochlear nucleus that may contribute to auditory hypersensitivity. RNA was isolated from cochlear nuclei of Fmr1-KO and WT mice. Using next-generation sequencing (RNA-seq), the transcriptomes of Fmr1-KO mice and WT mice (n = 3 each) were compared and analyzed using gene ontology programs. We identified 270 unique, differentially expressed genes between Fmr1-KO and WT cochlear nuclei. Upregulated genes (67%) are enriched in those encoding secreted molecules. Downregulated genes (33%) are enriched in neuronal function, including synaptic pathways, some of which are ideal candidate genes that may contribute to hyperacusis. The loss of FMRP can affect the expression of genes in the cochlear nucleus that are important for neuronal signaling. One of these, Kcnab2, which encodes a subunit of the Shaker voltage-gated potassium channel, is expressed at an abnormally low level in the Fmr1-KO cochlear nucleus. Kcnab2 and other differentially expressed genes may represent pathways for the development of hyperacusis. Future studies will be aimed at investigating the effects of these altered genes on hyperacusis. N/A Laryngoscope, 134:1363-1371, 2024.

Cell-type-specific disruption of cortico-striatal circuitry drives repetitive patterns of behavior in fragile X syndrome model mice

Individuals with fragile X syndrome (FXS) are frequently diagnosed with autism spectrum disorder (ASD), including increased risk for restricted and repetitive behaviors (RRBs). Consistent with observations in humans, FXS model mice display distinct RRBs and hyperactivity that are consistent with dysfunctional cortico-striatal circuits, an area relatively unexplored in FXS. Using a multidisciplinary approach, we dissect the contribution of two populations of striatal medium spiny neurons (SPNs) in the expression of RRBs in FXS model mice. Here, we report that dysregulated protein synthesis at cortico-striatal synapses is a molecular culprit of the synaptic and ASD-associated motor phenotypes displayed by FXS model mice. Cell-type-specific translational profiling of the FXS mouse striatum reveals differentially translated mRNAs, providing critical information concerning potential therapeutic targets. Our findings uncover a cell-type-specific impact of the loss of fragile X messenger ribonucleoprotein (FMRP) on translation and the sequence of neuronal events in the striatum that drive RRBs in FXS.

Prolonged and specific spatial training during adolescence reverses adult hippocampal network impairments in a mouse model of fragile X syndrome

The fragile X syndrome (FXS) is the leading monogenetic cause of cognitive impairment and autism. A hallmark of FXS in patients and the FXS mouse model (Fmr1 KO) is an overabundance of immature appearing dendritic spines in the cortex and hippocampus which is associated with behavioral deficits. Spine analysis in the different hippocampal subregions and at different developmental stages revealed that in adult mice, hippocampal spine pathology occurs specifically in the CA3 subregion, which plays a pivotal role in pattern completion processes important for efficient memory recall from parts of the initial memory stimulus. In line with this synaptic defect we document an impairment in memory recall during partially cued reference memory test in the Morris water maze task. This is accompanied by impaired recruitment of engram cells as well as impaired spine structural plasticity in the CA3 region. In order to promote hippocampal network development adolescent mice were either raised in an enriched environment or subjected to specific hippocampus-dependent spatial training. Intriguingly, only specific spatial training alleviated the cognitive symptoms and the spine phenotype shown in adult Fmr1 KO mice suggesting that specific stimulation of hippocampal networks during development might be used in the future as a therapeutic strategy.

Early Administration of the Phytocannabinoid Cannabidivarin Prevents the Neurobehavioral Abnormalities Associated with the Fmr1-KO Mouse Model of Fragile X Syndrome.

Phytocannabinoids, including the non-addictive cannabis component cannabidivarin (CBDV), have been reported to hold therapeutic potential in several neurodevelopmental disorders (NDDs). Nonetheless, the therapeutic value of phytocannabinoids for treating Fragile X syndrome (FXS), a major NDD, remains unexplored. Here, we characterized the neurobehavioral effects of CBDV at doses of 20 or 100 mg/kg in the Fmr1-knockout (Fmr1-KO) mouse model of FXS using two temporally different intraperitoneal regimens: subchronic 10-day delivery during adulthood (Study 1: rescue treatment) or chronic 5-week delivery at adolescence (Study 2: preventive treatment). Behavioral tests assessing FXS-like abnormalities included anxiety, locomotor, cognitive, social and sensory alterations. Expression of inflammatory and plasticity markers was investigated in the hippocampus and prefrontal cortex. When administered during adulthood (Study 1), the effects of CBDV were marginal, rescuing at the lower dose only the acoustic hyper-responsiveness of Fmr1-KO mice and at both doses their altered hippocampal expression of neurotrophins. When administered during adolescence (Study 2), CBDV at both doses prevented the cognitive, social and acoustic alterations of adult Fmr1-KO mice and modified the expression of several inflammatory brain markers in both wild-type littermates and mutants. These findings warrant the therapeutic potential of CBDV for preventing neurobehavioral alterations associated with FXS, highlighting the relevance of its early administration.

Hyperacusis in the Adult Fmr1-KO Mouse Model of Fragile X Syndrome: The Therapeutic Relevance of Cochlear Alterations and BKCa Channels.

Hyperacusis, i.e., an increased sensitivity to sounds, is described in several neurodevelopmental disorders (NDDs), including Fragile X Syndrome (FXS). The mechanisms underlying hyperacusis in FXS are still largely unknown and effective therapies are lacking. Big conductance calcium-activated potassium (BKCa) channels were proposed as a therapeutic target to treat several behavioral disturbances in FXS preclinical models, but their role in mediating their auditory alterations was not specifically addressed. Furthermore, studies on the acoustic phenotypes of FXS animal models mostly focused on central rather than peripheral auditory pathways. Here, we provided an extensive characterization of the peripheral auditory phenotype of the Fmr1-knockout (KO) mouse model of FXS at adulthood. We also assessed whether the acute administration of Chlorzoxazone, a BKCa agonist, could rescue the auditory abnormalities of adult mutant mice. Fmr1-KO mice both at 3 and 6 months showed a hyperacusis-like startle phenotype with paradoxically reduced auditory brainstem responses associated with a loss of ribbon synapses in the inner hair cells (IHCs) compared to their wild-type (WT) littermates. BKCa expression was markedly reduced in the IHCs of KOs compared to WT mice, but only at 6 months, when Chlorzoxazone rescued mutant auditory dysfunction. Our findings highlight the age-dependent and progressive contribution of peripheral mechanisms and BKCa channels to adult hyperacusis in FXS, suggesting a novel therapeutic target to treat auditory dysfunction in NDDs.

Novel presynaptic assay system revealed that metformin ameliorates exaggerated synaptic release and Munc18-1 accumulation in presynapses of neurons from Fragile X syndrome mouse model

Fragile X syndrome (FXS) is a developmental disorder characterized by intellectual disability and autistic-like behaviors. These symptoms are supposed to result from dysregulated translation in pre- and postsynapses, resulting in aberrant synaptic plasticity. Although most drug development research on FXS has focused on aberrant postsynaptic functions by excess translation in postsynapses, the effect of drug candidates on FXS in presynaptic release is largely unclear. In this report, we developed a novel assay system using neuron ball culture with beads to induce presynapse formation, allowing for the analysis of presynaptic phenotypes, including presynaptic release. Metformin, which is shown to rescue core phenotypes in FXS mouse model by normalizing dysregulated translation, ameliorated the exaggerated presynaptic release of neurons of FXS model mouse using this assay system. Furthermore, metformin suppressed the excess accumulation of the active zone protein Munc18-1, which is supposed to be locally translated in presynapses. These results suggest that metformin rescues both postsynaptic and presynaptic phenotypes by inhibiting excess translation in FXS neurons.

Altered striatal actin dynamics drives behavioral inflexibility in a mouse model of fragile X syndrome

The proteome of glutamatergic synapses is diverse across the mammalian brain and involved in neurodevelopmental disorders (NDDs). Among those is fragile X syndrome (FXS), an NDD caused by the absence of the functional RNA-binding protein FMRP. Here, we demonstrate how the brain region-specific composition of postsynaptic density (PSD) contributes to FXS. In the striatum, the FXS mouse model shows an altered association of the PSD with the actin cytoskeleton, reflecting immature dendritic spine morphology and reduced synaptic actin dynamics. Enhancing actin turnover with constitutively active RAC1 ameliorates these deficits. At the behavioral level, the FXS model displays striatal-driven inflexibility, a typical feature of FXS individuals, which is rescued by exogenous RAC1. Striatal ablation of Fmr1 is sufficient to recapitulate behavioral impairments observed in the FXS model. These results indicate that dysregulation of synaptic actin dynamics in the striatum, a region largely unexplored in FXS, contributes to the manifestation of FXS behavioral phenotypes.

Acute and Repeated Administration of NLX-101, a Selective Serotonin-1A Receptor Biased Agonist, Reduces Audiogenic Seizures in Developing Fmr1 Knockout Mice

Fragile XSyndrome (FXS) is a leading known genetic cause of Autism Spectrum Disorders (ASD) and intellectual disability. A consistent and debilitating phenotype of FXS is sensory hypersensitivity that manifests strongly in the auditory domain and may lead to delayed language and high anxiety. The mouse model of FXS, the Fmr1 KO mouse, also shows auditory hypersensitivity, an extreme form of which is seen as audiogenic seizures (AGS). The midbrain inferior colliculus (IC) is critically involved in generating audiogenic seizures and IC neurons are hyper-responsive to sounds in developing Fmr1 KO mice. Serotonin-1A receptor (5-HT1A) activation reduces IC activity. Therefore, we tested whether 5-HT1A activation is sufficient to reduce audiogenic seizures in Fmr1 KO mice. A selective and post-synaptic 5-HT1A receptor biased agonist, 3-Chloro-4-fluorophenyl-[4-fluoro-4-[[(5-methylpyrimidin-2-ylmethyl)amino]methyl]piperidin-1-yl] methanone (NLX-101, 0.6, 1.2, 1.8 or 2.4 mg/kg, i.p.) was administered to Fmr1 KO mice 15 min before seizure induction. Whereas the 0.6 mg/kg dose was ineffective in reducing seizures, the 1.2, 1.8 and 2.4 mg/kg doses of NLX-101 dramatically reduced seizures and increased mouse survival. Treatment with a combination of NLX-101 and 5-HT1A receptor antagonists prevented the protective effects of NLX-101, indicating that NLX-101 acts selectively through 5-HT1A receptors to reduce audiogenic seizures. NLX-101 (1.8 mg/kg) was still strongly effective in reducing seizures even after repeated administration over 5 days, suggesting an absence of tachyphylaxis to the effects of the compound. Together, these studies point to a promising treatment option targeting post-synaptic 5-HT1A receptors to reduce auditory hypersensitivity in FXS, and potentially across autism spectrum disorders.

The inhibition of NCS-1 binding to Ric8a rescues fragile X syndrome mice model phenotypes

Fragile X syndrome (FXS) is caused by the loss of function of Fragile X mental retardation protein (FMRP). FXS is one of the leading monogenic causes of intellectual disability (ID) and autism. Although it is caused by the failure of a single gene, FMRP that functions as an RNA binding protein affects a large number of genes secondarily. All these genes represent hundreds of potential targets and different mechanisms that account for multiple pathological features, thereby hampering the search for effective treatments. In this scenario, it seems desirable to reorient therapies toward more general approaches. Neuronal calcium sensor 1 (NCS-1), through its interaction with the guanine-exchange factor Ric8a, regulates the number of synapses and the probability of the release of a neurotransmitter, the two neuronal features that are altered in FXS and other neurodevelopmental disorders. Inhibitors of the NCS-1/Ric8a complex have been shown to be effective in restoring abnormally high synapse numbers as well as improving associative learning in FMRP mutant flies. Here, we demonstrate that phenothiazine FD44, an NCS-1/Ric8a inhibitor, has strong inhibition ability in situ and sufficient bioavailability in the mouse brain. More importantly, administration of FD44 to two different FXS mouse models restores well-known FXS phenotypes, such as hyperactivity, associative learning, aggressive behavior, stereotype, or impaired social approach. It has been suggested that dopamine (DA) may play a relevant role in the behavior and in neurodevelopmental disorders in general. We have measured DA and its metabolites in different brain regions, finding a higher metabolic rate in the limbic area, which is also restored with FD44 treatment. Therefore, in addition to confirming that the NCS-1/Ric8a complex is an excellent therapeutic target, we demonstrate the rescue effect of its inhibitor on the behavior of cognitive and autistic FXS mice and show DA metabolism as a FXS biochemical disease marker.

Long-term behavioral effects of prenatal stress in the Fmr1-knock-out mouse model for fragile X syndrome.

Fragile X syndrome (FXS) is a major neurodevelopmental disorder and the most common monogenic cause of autism spectrum disorder (ASD). FXS is caused by a mutation in the X-linked FMR1 gene leading to the absence of the FMRP protein, inducing several behavioral deficits, including motor, emotional, cognitive, and social abnormalities. Beside its clear genetic origins, FXS can be modulated by environmental factors, e.g., stress exposure: indeed the behavioral phenotype of FXS, as well as of ASD patients can be exacerbated by the repeated experience of stressful events, especially early in life. Here we investigated the long-term effects of prenatal exposure to unpredictable chronic stress on the behavioral phenotype of the Fmr1-knock-out (KO) mouse model for FXS and ASD. Mice were tested for FXS- and ASD-relevant behaviors first at adulthood (3 months) and then at aging (18 months), in order to assess the persistence and the potential time-related progression of the stress effects. Stress induced the selective emergence of behavioral deficits in Fmr1-KO mice that were evident in spatial memory only at aging. Stress also exerted several age-specific behavioral effects in mice of both genotypes: at adulthood it enhanced anxiety levels and reduced social interaction, while at aging it enhanced locomotor activity and reduced the complexity of ultrasonic calls. Our findings underline the relevance of gene-environment interactions in mouse models of neurodevelopmental syndromes and highlight the long-term behavioral impact of prenatal stress in laboratory mice.