Distributed X chromosome inactivation in brain circuitry is associated with X-linked disease penetrance of behavior.

The precise anatomical degree of brain X chromosome inactivation (XCI) that is sufficient to alter X-linked disorders in females is unclear. Here, we quantify whole-brain XCI at single-cell resolution to discover a prevalent activation ratio of maternal to paternal X at 60:40 across all divisions of the adult brain. This modest, non-random XCI influences X-linked disease penetrance: maternal transmission of the fragile X mental retardation 1 (Fmr1)-knockout (KO) allele confers 55% of total brain cells with mutant X-active, which is sufficient for behavioral penetrance, while 40% produced from paternal transmission is tolerated. Local XCI mosaicism within affected maternal Fmr1-KO mice further specifies sensorimotor versus social anxiety phenotypes depending on which distinct brain circuitry is most affected, with only a 50%-55% mutant X-active threshold determining penetrance. Thus, our results define a model of X-linked disease penetrance in females whereby distributed XCI among single cells populating brain circuitries can regulate the behavioral penetrance of an X-linked mutation.

LLPS of FXR proteins drives replication organelle clustering for ß-coronaviral proliferation.

ß-Coronaviruses remodel host endomembranes to form double-membrane vesicles (DMVs) as replication organelles (ROs) that provide a shielded microenvironment for viral RNA synthesis in infected cells. DMVs are clustered, but the molecular underpinnings and pathophysiological functions remain unknown. Here, we reveal that host fragile X-related (FXR) family proteins (FXR1/FXR2/FMR1) are required for DMV clustering induced by expression of viral non-structural proteins (Nsps) Nsp3 and Nsp4. Depleting FXRs results in DMV dispersion in the cytoplasm. FXR1/2 and FMR1 are recruited to DMV sites via specific interaction with Nsp3. FXRs form condensates driven by liquid-liquid phase separation, which is required for DMV clustering. FXR1 liquid droplets concentrate Nsp3 and Nsp3-decorated liposomes in vitro. FXR droplets facilitate recruitment of translation machinery for efficient translation surrounding DMVs. In cells depleted of FXRs, SARS-CoV-2 replication is significantly attenuated. Thus, SARS-CoV-2 exploits host FXR proteins to cluster viral DMVs via phase separation for efficient viral replication.

Proteomics insights into fragile X syndrome: Unraveling molecular mechanisms and therapeutic avenues.

Fragile X Syndrome (FXS) is a neurodevelopment disorder characterized by cognitive impairment, behavioral challenges, and synaptic abnormalities, with a genetic basis linked to a mutation in the FMR1 (Fragile X Messenger Ribonucleoprotein 1) gene that results in a deficiency or absence of its protein product, Fragile X Messenger Ribonucleoprotein (FMRP). In recent years, mass spectrometry (MS) - based proteomics has emerged as a powerful tool to uncover the complex molecular landscape underlying FXS. This review provides a comprehensive overview of the proteomics studies focused on FXS, summarizing key findings with an emphasis on dysregulated proteins associated with FXS. These proteins span a wide range of cellular functions including, but not limited to, synaptic plasticity, RNA translation, and mitochondrial function. The work conducted in these proteomic studies provides a more holistic understanding to the molecular pathways involved in FXS and considerably enhances our knowledge into the synaptic dysfunction seen in FXS.

KIF5B plays important roles in dendritic spine plasticity and dendritic localization of PSD95 and FMRP in the mouse cortex in vivo.

Kinesin 1 (KIF5) is one major type of motor protein in neurons, but its members' function in the intact brain remains less studied. Using in vivo two-photon imaging, we find that conditional knockout of Kif5b (KIF5B cKO) in CaMKIIα-Cre-expressing neurons shows heightened turnover and lower stability of dendritic spines in layer 2/3 pyramidal neurons with reduced spine postsynaptic density protein 95 acquisition in the mouse cortex. Furthermore, the RNA-binding protein fragile X mental retardation protein (FMRP) is translocated to the proximity of newly formed spines several hours before the spine formation events in vivo in control mice, but this preceding transport of FMRP is abolished in KIF5B cKO mice. We further find that FMRP is localized closer to newly formed spines after fear extinction, but this learning-dependent localization is disrupted in KIF5B cKO mice. Our findings provide the crucial in vivo evidence that KIF5B is involved in the dendritic targeting of synaptic proteins that underlies dendritic spine plasticity.

Variation of FMRP Expression in Peripheral Blood Mononuclear Cells from Individuals with Fragile X Syndrome.

Fragile X syndrome (FXS) is the most common heritable cause of intellectual disability and autism spectrum disorder. The syndrome is often caused by greatly reduced or absent protein expression from the fragile X messenger ribonucleoprotein 1 (FMR1) gene due to expansion of a 5'-non-coding trinucleotide (CGG) element beyond 200 repeats (full mutation). To better understand the complex relationships among FMR1 allelotype, methylation status, mRNA expression, and FMR1 protein (FMRP) levels, FMRP was quantified in peripheral blood mononuclear cells for a large cohort of FXS (n = 154) and control (n = 139) individuals using time-resolved fluorescence resonance energy transfer. Considerable size and methylation mosaicism were observed among individuals with FXS, with FMRP detected only in the presence of such mosaicism. No sample with a minimum allele size greater than 273 CGG repeats had significant levels of FMRP. Additionally, an association was observed between FMR1 mRNA and FMRP levels in FXS samples, predominantly driven by those with the lowest FMRP values. This study underscores the complexity of FMR1 allelotypes and FMRP expression and prompts a reevaluation of FXS therapies aimed at reactivating large full mutation alleles that are likely not capable of producing sufficient FMRP to improve cognitive function.

Challenges in developing therapies in fragile X syndrome: how the FXLEARN trial can guide research.

Fragile X syndrome (FXS), the most common inherited cause of intellectual disability and the single-gene cause of autism, is caused by decreased expression of the fragile X messenger ribonucleoprotein protein (FMRP), a ribosomal-associated RNA-binding protein involved in translational repression. Extensive preclinical work in several FXS animal models supported the therapeutic potential of decreasing metabotropic glutamate receptor (mGluR) signaling to correct translation of proteins related to synaptic plasticity; however, multiple clinical trials failed to show conclusive evidence of efficacy. In this issue of the JCI, Berry-Kravis and colleagues conducted the FXLEARN clinical trial to address experimental design concerns from previous trials. Unfortunately, despite treatment of young children with combined pharmacological and learning interventions for a prolonged period, no efficacy of blocking mGluR activity was observed. Future systematic evaluation of potential therapeutic approaches should evaluate consistency between human and animal pathophysiological mechanisms, utilize innovative clinical trial design from FXLEARN, and incorporate translatable biomarkers.

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.

BREACHing new grounds in fragile X syndrome: Trinucleotide expansion linked to genome-wide heterochromatin domains and genome misfolding.

In a recent study in Cell, Malachowski et al.1 show that the trinucleotide expansion in the FMR1 gene underlying fragile X syndrome triggers formation of large heterochromatin domains across the genome, resulting in the repression of synaptic genes housed within these domains.

Bisphenol F affects neurodevelopmental gene expression, mushroom body development, and behavior in Drosophila melanogaster.

Bisphenol F (BPF) is a potential neurotoxicant used as a replacement for bisphenol A (BPA) in polycarbonate plastics and epoxy resins. We investigated the neurodevelopmental impacts of BPF exposure using Drosophila melanogaster as a model. Our transcriptomic analysis indicated that developmental exposure to BPF caused the downregulation of neurodevelopmentally relevant genes, including those associated with synapse formation and neuronal projection. To investigate the functional outcome of BPF exposure, we evaluated neurodevelopmental impacts across two genetic strains of Drosophila- w1118 (control) and the Fragile X Syndrome (FXS) model-by examining both behavioral and neuronal phenotypes. We found that BPF exposure in w1118 Drosophila caused hypoactive larval locomotor activity, decreased time spent grooming by adults, reduced courtship activity, and increased the severity but not frequency of ß-lobe midline crossing defects by axons in the mushroom body. In contrast, although BPF reduced peristaltic contractions in FXS larvae, it had no impact on other larval locomotor phenotypes, grooming activity, or courtship activity. Strikingly, BPF exposure reduced both the severity and frequency of ß-lobe midline crossing defects in the mushroom body of FXS flies, a phenotype previously observed in FXS flies exposed to BPA. This data indicates that BPF can affect neurodevelopment and its impacts vary depending on genetic background. Further, BPF may elicit a gene-environment interaction with Drosophila fragile X messenger ribonucleoprotein 1 (dFmr1)-the ortholog of human FMR1, which causes fragile X syndrome and is the most common monogenetic cause of intellectual disability and autism spectrum disorder.

Circuit-based intervention corrects excessive dentate gyrus output in the fragile X mouse model.

Abnormal cellular and circuit excitability is believed to drive many core phenotypes in fragile X syndrome (FXS). The dentate gyrus is a brain area performing critical computations essential for learning and memory. However, little is known about dentate circuit defects and their mechanisms in FXS. Understanding dentate circuit dysfunction in FXS has been complicated by the presence of two types of excitatory neurons, the granule cells and mossy cells. Here we report that loss of FMRP markedly decreased excitability of dentate mossy cells, a change opposite to all other known excitability defects in excitatory neurons in FXS. This mossy cell hypo-excitability is caused by increased Kv7 function in Fmr1 knockout (KO) mice. By reducing the excitatory drive onto local hilar interneurons, hypo-excitability of mossy cells results in increased excitation/inhibition ratio in granule cells and thus paradoxically leads to excessive dentate output. Circuit-wide inhibition of Kv7 channels in Fmr1 KO mice increases inhibitory drive onto granule cells and normalizes the dentate output in response to physiologically relevant theta-gamma coupling stimulation. Our study suggests that circuit-based interventions may provide a promising strategy in this disorder to bypass irreconcilable excitability defects in different cell types and restore their pathophysiological consequences at the circuit level.

The NSP3 protein of SARS-CoV-2 binds fragile X mental retardation proteins to disrupt UBAP2L interactions.

Viruses interact with numerous host factors to facilitate viral replication and to dampen antiviral defense mechanisms. We currently have a limited mechanistic understanding of how SARS-CoV-2 binds host factors and the functional role of these interactions. Here, we uncover a novel interaction between the viral NSP3 protein and the fragile X mental retardation proteins (FMRPs: FMR1, FXR1-2). SARS-CoV-2 NSP3 mutant viruses preventing FMRP binding have attenuated replication in vitro and reduced levels of viral antigen in lungs during the early stages of infection. We show that a unique peptide motif in NSP3 binds directly to the two central KH domains of FMRPs and that this interaction is disrupted by the I304N mutation found in a patient with fragile X syndrome. NSP3 binding to FMRPs disrupts their interaction with the stress granule component UBAP2L through direct competition with a peptide motif in UBAP2L to prevent FMRP incorporation into stress granules. Collectively, our results provide novel insight into how SARS-CoV-2 hijacks host cell proteins and provides molecular insight into the possible underlying molecular defects in fragile X syndrome.

The multifaceted role of Fragile X-Related Protein 1 (FXR1) in cellular processes: an updated review on cancer and clinical applications.

RNA-binding proteins (RBPs) modulate the expression level of several target RNAs (such as mRNAs) post-transcriptionally through interactions with unique binding sites in the 3'-untranslated region. There is mounting information that suggests RBP dysregulation plays a significant role in carcinogenesis. However, the function of FMR1 autosomal homolog 1(FXR1) in malignancies is just beginning to be unveiled. Due to the diversity of their RNA-binding domains and functional adaptability, FXR1 can regulate diverse transcript processing. Changes in FXR1 interaction with RNA networks have been linked to the emergence of cancer, although the theoretical framework defining these alterations in interaction is insufficient. Alteration in FXR1 expression or localization has been linked to the mRNAs of cancer suppressor genes, cancer-causing genes, and genes involved in genomic expression stability. In particular, FXR1-mediated gene regulation involves in several cellular phenomena related to cancer growth, metastasis, epithelial-mesenchymal transition, senescence, apoptosis, and angiogenesis. FXR1 dysregulation has been implicated in diverse cancer types, suggesting its diagnostic and therapeutic potential. However, the molecular mechanisms and biological effects of FXR1 regulation in cancer have yet to be understood. This review highlights the current knowledge of FXR1 expression and function in various cancer situations, emphasizing its functional variety and complexity. We further address the challenges and opportunities of targeting FXR1 for cancer diagnosis and treatment and propose future directions for FXR1 research in oncology. This work intends to provide an in-depth review of FXR1 as an emerging oncotarget with multiple roles and implications in cancer biology and therapy.

Altered brain serotonin 5-HT1A receptor expression and function in juvenile Fmr1 knockout mice.

There are no approved pharmacotherapies for fragile X syndrome (FXS), a rare neurodevelopmental disorder caused by a mutation in the FMR1 promoter region that leads to various symptoms, including intellectual disability and auditory hypersensitivity. The gene that encodes inhibitory serotonin 1A receptors (5-HT1ARs) is differentially expressed in embryonic brain tissue from individuals with FXS, and 5-HT1ARs are highly expressed in neural systems that are disordered in FXS, providing a rationale to focus on 5-HT1ARs as targets to treat symptoms of FXS. We examined agonist-labeled 5-HT1AR densities in male and female Fmr1 knockout mice and found no differences in whole-brain 5-HT1AR expression in adult control compared to Fmr1 knockout mice. However, juvenile Fmr1 knockout mice had lower whole-brain 5-HT1AR expression than age-matched controls. Consistent with these results, juvenile Fmr1 knockout mice showed reduced behavioral responses elicited by the 5-HT1AR agonist (R)-8-OH-DPAT, effects blocked by the selective 5-HT1AR antagonist, WAY-100635. Also, treatment with the selective 5-HT1AR agonist, NLX-112, dose-dependently prevented audiogenic seizures (AGS) in juvenile Fmr1 knockout mice, an effect reversed by WAY-100635. Suggestive of a potential role for 5-HT1ARs in regulating AGS, compared to males, female Fmr1 knockout mice had a lower prevalence of AGS and higher expression of antagonist-labeled 5-HT1ARs in the inferior colliculus and auditory cortex. These results provide preclinical support that 5-HT1AR agonists may be therapeutic for young individuals with FXS hypersensitive to auditory stimuli.

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

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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. LEVEL OF EVIDENCE: N/A Laryngoscope, 134:1363-1371, 2024.

Intellectual Disability and Behavioral Deficits Linked to CYFIP1 Missense Variants Disrupting Actin Polymerization.

BACKGROUND: 15q11.2 deletions and duplications have been linked to autism spectrum disorder, schizophrenia, and intellectual disability. Recent evidence suggests that dysfunctional CYFIP1 (cytoplasmic FMR1 interacting protein 1) contributes to the clinical phenotypes observed in individuals with 15q11.2 deletion/duplication syndrome. CYFIP1 plays crucial roles in neuronal development and brain connectivity, promoting actin polymerization and regulating local protein synthesis. However, information about the impact of single nucleotide variants in CYFIP1 on neurodevelopmental disorders is limited. METHODS: Here, we report a family with 2 probands exhibiting intellectual disability, autism spectrum disorder, spastic tetraparesis, and brain morphology defects and who carry biallelic missense point mutations in the CYFIP1 gene. We used skin fibroblasts from one of the probands, the parents, and typically developing individuals to investigate the effect of the variants on the functionality of CYFIP1. In addition, we generated Drosophila knockin mutants to address the effect of the variants in vivo and gain insight into the molecular mechanism that underlies the clinical phenotype. RESULTS: Our study revealed that the 2 missense variants are in protein domains responsible for maintaining the interaction within the wave regulatory complex. Molecular and cellular analyses in skin fibroblasts from one proband showed deficits in actin polymerization. The fly model for these mutations exhibited abnormal brain morphology and F-actin loss and recapitulated the core behavioral symptoms, such as deficits in social interaction and motor coordination. CONCLUSIONS: Our findings suggest that the 2 CYFIP1 variants contribute to the clinical phenotype in the probands that reflects deficits in actin-mediated brain development processes.

Enrichment of the Local Synaptic Translatome for Genetic Risk Associated With Schizophrenia and Autism Spectrum Disorder.

BACKGROUND: Genes that encode synaptic proteins or messenger RNA targets of the RNA-binding protein FMRP (fragile X messenger ribonucleoprotein) have been linked to schizophrenia and autism spectrum disorder (ASD) through the enrichment of genetic variants that confer risk for these disorders. FMRP binds many transcripts with synaptic functions and is thought to regulate their local translation, a process that enables rapid and compartmentalized protein synthesis required for development and plasticity. METHODS: We used summary statistics from large-scale genome-wide association studies of schizophrenia (74,776 cases, 101,023 controls) and ASD (18,381 cases, 27,969 controls) to test the hypothesis that the subset of synaptic genes that encode localized transcripts is more strongly associated with each disorder than nonlocalized transcripts. We also postulated that this subset of synaptic genes is responsible for associations attributed to FMRP targets. RESULTS: Schizophrenia associations were enriched in genes encoding localized synaptic transcripts compared to the remaining synaptic genes or to the remaining localized transcripts; this also applied to ASD associations, although only for transcripts observed after stimulation by fear conditioning. The genetic associations with either disorder captured by these gene sets were independent of those derived from FMRP targets. Schizophrenia association was related to FMRP interactions with messenger RNAs in somata, but not in dendrites, while ASD association was related to FMRP binding in either compartment. CONCLUSIONS: Our data suggest that synaptic transcripts capable of local translation are particularly relevant to the pathogenesis of schizophrenia and ASD, but they do not characterize the associations attributed to current sets of FMRP targets.

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.

Structural Dynamics Role of AGG Interruptions in Inhibition CGG Repeat Expansion Associated with Fragile X Syndrome.

Abnormal expansion of trinucleotide CGG repeats is responsible for Fragile X syndrome. AGG interruptions in CGG repeat tracts were found in most healthy individuals, suggesting a crucial role in preventing disease-prone repeat expansion. Previous biophysics studies emphasize a difference in the secondary structure affected by AGG interruptions. However, the mechanism of how AGG interruptions impede repeat expansion remains elusive. We utilized single-molecule fluorescence resonance energy transfer spectroscopy to investigate the structural dynamics of CGG repeats and their AGG-interrupted variants. Tandem CGG repeats fold into a stem-loop hairpin structure with the capability to undergo a conformational rearrangement to modulate the length of the overhang. However, this conformational rearrangement is much more retarded when two AGG interruptions are present. Considering the significance of hairpin slippage in repeat expansion, we present a molecular basis suggesting that the internal loop created by two AGG interruptions acts as a barrier, obstructing the hairpin slippage reconfiguration. This impediment potentially plays a crucial role in curbing abnormal expansion, thereby contributing to the genomic stability.

Complex interplay between FMRP and DHX9 during DNA replication stress.

Mutations in, or deficiency of, fragile X messenger ribonucleoprotein (FMRP) is responsible for the Fragile X syndrome (FXS), the most common cause for inherited intellectual disability. FMRP is a nucleocytoplasmic protein, primarily characterized as a translation repressor with poorly understood nuclear function(s). We recently reported that FXS patient cells lacking FMRP sustain higher level of DNA double-strand breaks (DSBs) than normal cells, specifically at sequences prone to forming R-loops, a phenotype further exacerbated by DNA replication stress. Moreover, expression of FMRP, and not an FMRPI304N mutant known to cause FXS, reduced R-loop-associated DSBs. We subsequently reported that recombinant FMRP directly binds R-loops, primarily through the carboxyl terminal intrinsically disordered region. Here, we show that FMRP directly interacts with an RNA helicase, DHX9. This interaction, which is mediated by the amino terminal structured domain of FMRP, is reduced with FMRPI304N. We also show that FMRP inhibits DHX9 helicase activity on RNA:DNA hybrids and the inhibition is also dependent on the amino terminus. Furthermore, the FMRPI304N mutation causes both FMRP and DHX9 to persist on the chromatin in replication stress. These results suggest an antagonistic relationship between FMRP and DHX9 at the chromatin, where their proper interaction leads to dissociation of both proteins from the fully resolved R-loop. We propose that the absence or the loss of function of FMRP leads to persistent presence of DHX9 or both proteins, respectively, on the unresolved R-loop, ultimately leading to DSBs. Our study sheds new light on our understanding of the genome functions of FMRP.

Cerebellar contribution to autism-relevant behaviors in fragile X syndrome models.

Cerebellar dysfunction has been linked to autism spectrum disorders (ASDs). Although cerebellar pathology has been observed in individuals with fragile X syndrome (FXS) and in mouse models of the disorder, a cerebellar functional contribution to ASD-relevant behaviors in FXS has yet to be fully characterized. In this study, we demonstrate a critical cerebellar role for Fmr1 (fragile X messenger ribonucleoprotein 1) in ASD-relevant behaviors. First, we identify reduced social behaviors, sensory hypersensitivity, and cerebellar dysfunction, with loss of cerebellar Fmr1. We then demonstrate that cerebellar-specific expression of Fmr1 is sufficient to impact social, sensory, cerebellar dysfunction, and cerebro-cortical hyperexcitability phenotypes observed in global Fmr1 mutants. Moreover, we demonstrate that targeting the ASD-implicated cerebellar region Crus1 ameliorates behaviors in both cerebellar-specific and global Fmr1 mutants. Together, these results demonstrate a critical role for the cerebellar contribution to FXS-related behaviors, with implications for future therapeutic strategies.