Fmr1 KO Research Articles (Page 1)

Background: Fragile X Syndrome (FXS) is the most common monogenic cause of autism spectrum disorders, and is characterized by the excessive immature excitatory synapses in cortical neurons, leading to excitatory/inhibitory imbalance and core autistic behaviors. This synaptic pathology has been attributed to dysregulated levels of synaptic proteins, including CYFIP2: a key regulator of synaptic structure and plasticity. However, the mechanism underlying the increased CYFIP2 protein level in FXS neurons remains unclear. Neurons abundantly secrete extracellular vesicles (EVs) enriched with bioactive cargos (proteins and miRNAs). Objectives: the goal of this research is to identify whether EV-dependent secretion plays important roles in regulating the intracellular CYFIP2 protein level in WT and FXS neurons. Methods and Results: our proteomic analysis reveals that CYFIP2 protein is packaged in EVs released by mouse cortical neurons. Pharmacological and genetic blockades of neuronal EV release significantly elevated intracellular CYFIP2 levels by 78 ± 14% and 168 ± 39%, respectively. Glutamate-evoked EV release significantly reduced the CYFIP2 level by 24 ± 2%. Neurons from Fmr1 KO mice, an FXS model, secreted significantly less EVs (46 ± 5%) than the wild type, and showed significantly elevated CYFIP2 (by 155 ± 31%). Evoking EV release in FXS neurons significantly lowered the intracellular CYFIP2 (by 53 ± 6%). Conclusions: these findings identify an EV-secretion-dependent mechanism that controls neuronal CYFIP2 level, implicating EV-mediated export in the regulation of synaptic protein homeostasis, synaptic remodeling, and FXS-associated synaptic deficits.

Loss of fragile X messenger ribonucleoprotein (FMRP) causes fragile X syndrome (FXS), an inherited neurodevelopmental disorder resulting in intellectual disability and autism spectrum disorder; however, the molecular function of FMRP remains uncertain. Here, using cell lines and fibroblasts and induced pluripotent stem cell-derived neurons from healthy individuals and patients with FXS, we showed that FMRP regulates collided ribosomes by recruiting activating signal cointegrator 1 complex subunit 3 (ASCC3), an early-acting ribosome-associated quality control (RQC) factor to collided ribosomes, and either positively or negatively regulating translation, depending on transcript context. Disease-associated ASCC3 variants that perturbed ASCC3-FMRP interaction were also found to be defective in ribosome association and handling of collided ribosomes. In cells of a patient with FXS and the Fmr1 KO mouse model, ASCC3 abundance was reduced, and overexpression of ASCC3 in the brains of fetal Fmr1 KO mice promoted neuronal migration. In addition, CRISPR-mediated activation of ASCC3 by lateral ventricular injection of adeno-associated virus (AAV) ameliorated synaptic defects and improved locomotor activity, cognitive deficits, obsessive-compulsive-like behavior, and social interaction deficits after 1 month in 2-month-old Fmr1 KO mice compared with untreated Fmr1 KO controls. In conclusion, these data implicated FMRP in the handling of collided ribosomes to maintain protein homeostasis during neurodevelopment and synaptogenesis and demonstrated proof of concept that targeting RQC may offer alternative treatment strategies for FXS.

Dysregulated spine morphology is a common feature in the pathology of many neurodevelopmental and neuropsychiatric disorders. Overabundant immature dendritic spines in the hippocampus are causally related to cognitive deficits of Fragile X syndrome (FXS), the most common form of heritable intellectual disability. Recent findings from us and others indicate autophagy plays important roles in synaptic stability and morphology, and autophagy is downregulated in FXS neurons. However, the mechanism remains unclear. In this study, we identified that activated autophagy degrades the eukaryotic initiation factor 4G1 (eIF4G1) and postsynaptic density protein-95 (PSD-95) in hippocampal neurons of Fmr1 KO mice and FXS neurons from patients, which subsequently corrected the dysregulated postsynaptic organization and actin assembly, the critical processes determining synaptic maturation and density. Centrally activating autophagy in hippocampus degrades eIF4G1 and PSD-95, restores actin dynamics, and improves cognition of Fmr1 KO mice. In human neurons derived from patients diagnosed with both FXS and intellectual disability, activating autophagy corrected the aberrant actin assembly. Thus, our findings revealed a previously unappreciated mechanism through which autophagy affects actin assembly and synaptic organization, suggesting a critical role of autophagy in regulating structural synaptic plasticity in healthy and diseased conditions.

The neurodevelopmental disorder fragile X syndrome (FXS) results from hypermethylation of the FMR1 gene, which prevents production of the FMRP protein. FMRP modulates the expression and function of a variety of proteins, including voltage-gated ion channels, such as hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels, which are integral to rhythmic activity in thalamic structures. Thalamocortical pathology, particularly involving the mediodorsal thalamus (MD), has been implicated in neurodevelopmental disorders such as FXS. MD connectivity with the medial prefrontal cortex (mPFC) is integral to executive functions such as working memory and social behaviours that are disrupted in FXS. We used a combination of retrograde labelling and ex vivo brain slice whole-cell electrophysiology in 40 wild-type and 42 Fmr1 knockout male mice to investigate how a lack of Fmr1 affects intrinsic cellular properties in lateral (MD-L) and medial (MD-M) MD neurons that project to the mPFC (MD→mPFC neurons). In MD-L neurons, Fmr1 knockout decreased the HCN-mediated membrane properties voltage sag and membrane after-hyperpolarization. We also identified a delay in rebound spike timing in both complex bursts and low-threshold spikes. In Fmr1 knockout mice, reduced HCN channel activity in MD-L→mPFC neurons impaired both the timing and the magnitude of HCN-mediated membrane potential regulation. Changes in response timing might adversely affect rhythm propagation in Fmr1 KO thalamocortical circuitry. MD thalamic neurons are crucial for maintaining rhythmic activity involved in cognitive and affective functions. Understanding specific mechanisms of thalamocortical circuit activity might lead to therapeutic interventions for individuals with FXS and other conditions characterized by thalamic dysrhythmia.

Clinical features of the fragile X syndrome (FXS) phenotype include intellectual disability, repetitive behaviors, social communication deficits, and, commonly, auditory hypersensitivity to acoustic stimuli. Electrophysiological studies have shown that FXS patients and Fmr1KO mice exhibit improper processing of auditory information in the cortical areas of the brain and the spiral ganglion of the cochlea. Synapses formed by spiral ganglion neurons on sensory hair cells (HC) are the first connection on the path that conveys the auditory information from the sensory cells to the brain. We confirmed the presence of fragile X mental retardation protein (FMRP) in the inner hair cells of the cochlea. Next, we analyzed the morphology of IHC ribbon synapses in early stages of postnatal development (P5, P14) and detected their delayed structural maturation in Fmr1 KO mice. Interestingly, the ultrastructure of inner hair cell ribbon synapses, studied by electron microscopy in adult mice (P48), has shown no specific dysmorphologies. Delayed structural maturation of presynaptic ribbons of auditory hair cells in Fmr1 KO mice may contribute to abnormal development of circuits induced by auditory experience.

While conventional synaptic plasticity (LTP and LTD) has been extensively examined in Fmr1 KO mice, evidence about the integrity of metaplasticity in these mice has been limited. This study provides a characterization of alterations in NMDA receptor mediated metaplasticity in the hippocampus in Fmr1 KO mice. The question of whether hippocampal LTP is altered in these mice remains unresolved, and changes in metaplasticity may partly explain the discrepancies across studies. Our findings not only identify novel synaptic phenotypes and their underlying mechanisms in the FXS mouse model, but also highlight potential therapeutic targets for FXS.

Cortical layer-specific abnormalities in auditory responses in a mouse model of Fragile X Syndrome.

Essential lipids enrich membrane-associated condensates to rescue synaptic morpho-functional deficits in a mouse model of autism.

Fragile X Syndrome (FXS) is the most common monogenetic cause of autism and inherited intellectual disability. A key feature of FXS symptomatology is altered sensory processing greatly affecting FXS individual’s life quality. Here, we use a combination of behavioral tests and slice physiology tools to study the neurophysiological alterations underlying aberrant sensory processing in the olfactory system of the FXS mouse model (Fmr1 KO). We focused on the piriform cortex (PC), since it is in this brain region where olfactory information is integrated and ultimately decoded. Using a go-no go behavioral task we have found that Fmr1 KO learn to discriminate between a rewarded and a not rewarded odorant but cannot distinguish complex odor mixtures, akin to what is found in the environment. Moreover, Fmr1 KO long-term memory is impaired compared to control mice suggesting possibly cortical processing alterations. In addition, electrophysiological data from PC layer II neurons of Fmr1 KO mice showed a hyperexcitable phenotype manifested by differences in active membrane properties and altered network connectivity. Taken together, our data suggest a possible causal link between the observed olfactory discrimination deficiencies in the Fmr1 KO mouse and the altered physiology of PC.

Predicting clinical therapeutic outcomes from preclinical animal studies remains an obstacle to developing treatments for neuropsychiatric disorders. Electrophysiological biomarkers analyzed consistently across species could bridge this divide. In humans, alpha oscillations in the resting state electroencephalogram (rsEEG) are altered in many disorders, but these disruptions have not yet been characterized in animal models. Here, we employ a uniform analytical method to show in males with fragile X syndrome (FXS) that the slowed alpha oscillations observed in adults are also present in children and in visual cortex of adult and juvenile Fmr1 -/y mice. We find that alpha-like oscillations in mice reflect the differential activity of two classes of inhibitory interneurons, but the phenotype is caused by deletion of Fmr1 specifically in cortical excitatory neurons. These results provide a framework for studying alpha oscillation disruptions across species, advance understanding of a critical rsEEG signature in the human brain and inform the cellular basis for a putative biomarker of FXS.

The brain serotonin (5-HT) system modulates glutamatergic and GABAergic transmission in almost every brain area, crucially regulating mood, food intake, body temperature, pain, hormone secretion, learning and memory. Previous studies suggest a disruption of the brain 5-HT system in Fragile X Syndrome, with abnormal activity of the 5-HT transporter leading to altered 5-HT brain levels. We provide an update on therapeutic effects exerted by drugs modulating serotonergic transmission on Fragile X patients and animal models. The enhancement of serotonergic transmission using Selective Serotonin Reuptake Inhibitors (SSRIs) corrected mood disorders and language deficits in Fragile X patients. In Fmr1 KO mice, a model of Fragile X Syndrome, selective 5-HT7 receptor agonists rescued synaptic plasticity, memory and stereotyped behavior. In addition, drugs specifically acting on 5-HT1A, 5-HT2 and 5-HT5 receptor subtypes were able to correct, respectively, epilepsy, learning deficits and hyperactivity in different Fragile X animal models. In conclusion, the SSRI treatment of Fragile X patients improves mood and language; in parallel, studies on animal models suggest that compounds selectively acting on distinct 5-HT receptor subtypes might provide a targeted correction of other Fragile X phenotypes, and thus should be further tested in clinical trials for future therapy.

Background:The neurodevelopmental disorder Fragile X syndrome (FXS) results from hypermethylation of the FMR1 gene which prevents FMRP production. FMRP modulates the expression and function of a wide variety of proteins, including voltage-gated ion channels such as Hyperpolarization-Activated Cyclic Nucleotide gated (HCN) channels, which are integral to rhythmic activity in thalamic structures. Thalamocortical pathology, particularly involving the mediodorsal thalamus (MD), has been implicated in neurodevelopmental disorders. MD connectivity with mPFC is integral to executive functions like working memory and social behaviors that are disrupted in FXS.Methods:We used a combination of retrograde labeling and ex vivo brain slice whole cell electrophysiology in 40 wild type and 42 Fmr1 KO male mice to investigate how a lack of Fmr1 affects intrinsic cellular properties in lateral (MD-L) and medial (MD-M) MD neurons that project to the medial prefrontal cortex (MD→mPFC neurons).Results:In MD-L neurons, Fmr1 knockout caused a decrease in HCN-mediated membrane properties such as voltage sag and membrane afterhyperpolarization. These changes in subthreshold properties were accompanied by changes in suprathreshold neuron properties such as the variability of action potential burst timing.Conclusions:In Fmr1 knockout mice, reduced HCN channel activity in MD→mPFC neurons impairs both the timing and magnitude of HCN-mediated membrane potential regulation. Changes in response timing may adversely affect rhythm propagation in Fmr1 KO thalamocortical circuitry. MD thalamic neurons are critical for maintaining rhythmic activity involved in cognitive and affective functions. Understanding specific mechanisms of thalamocortical circuit activity may lead to therapeutic interventions for individuals with FXS.

Electroencephalographic (EEG) recordings in individuals with Fragile X Syndrome (FXS) and the mouse model of FXS ( Fmr1 KO) display cortical hyperexcitability at rest, as well as deficits in sensory-driven cortical network synchrony. A form of circuit hyperexcitability is observed in ex vivo cortical slices of Fmr1 KO mice as prolonged persistent activity, or Up, states. It is unknown if the circuit mechanisms that cause prolonged Up states contribute to FXS-relevant EEG phenotypes. Here we examined the role of endocannabinoids (eCB) in prolonged Up states in slices and resting and sensory-driven EEG phenotypes in awake Fmr1 KO mice. Bidirectional changes in eCB function are reported in the Fmr1 KO that depend on synapse type (excitatory or inhibitory). We demonstrate that pharmacological or genetic reduction of Cannabinoid Receptor 1 (CB1R) in GABAergic neurons rescues prolonged cortical Up states and deficits in sensory-driven cortical synchrony in Fmr1 KO mice. In support of these findings, recordings from Fmr1 KO cortical Layer (L) 2/3 pyramidal neurons revealed enhanced CB1R-mediated suppression of inhibitory synaptic currents. In contrast, genetic reduction of Cnr1 in glutamatergic neurons did not affect Up state duration, but deletion of Fmr1 in the same neurons was sufficient to cause long Up states. These findings support a model where loss of Fmr1 in glutamatergic neurons leads to enhanced CB1R-mediated suppression of GABAergic synaptic transmission, prolonged cortical circuit activation and reduced sensory-driven circuit synchronization. Results suggest that antagonism of CB1Rs as a therapeutic strategy to correct sensory processing deficits in FXS.

Daily oscillation of the excitation/inhibition ratio is disrupted in two mouse models of autism

Mouse models of Fragile X Syndrome (FXS) have demonstrated impairments in excitatory and inhibitory sensory-evoked neuronal firing. Homeostatic plasticity, which encompasses a set of mechanisms to stabilize baseline activity levels, does not compensate for these changes in activity. Previous work has shown that impairments in homeostatic plasticity are observed in FXS, including deficits in synaptic scaling and intrinsic excitability. Here, we aimed to examine how homeostatic plasticity is altered in vivo in an Fmr1 KO mouse model following unilateral whisker deprivation (WD). We show that WD in the wild type leads to an increase in the proportion of L5/6 somatosensory neurons that are recruited, but this does not occur in the KO. In addition, we observed a change in the threshold of excitatory neurons at a later developmental stage in the KO. Compromised homeostatic plasticity in development could influence sensory processing and long-term cortical organization.

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.

IntroductionFragile X syndrome is an inherited X-linked disorder associated with intellectual disabilities that begin in childhood and last a lifetime. The symptoms overlap with autism spectrum disorder, and the syndrome predominantly affects males. Consequently, FXS research tends to favor analysis of social behaviors in males, leaving a gap in our understanding of other behavioral traits, especially in females.MethodsWe used a mouse model of FXS to analyze developmental, behavioral, neurochemical, and transcriptomic profiles in males and females.ResultsOur behavioral assays demonstrated locomotor hyperactivity, motor impulsivity, increased “approach” behavior in an approach-avoidance assay, and deficits in nest building behavior. Analysis of brain neurotransmitter content revealed deficits in striatal GABA, glutamate, and serotonin content. RNA sequencing of the ventral striatum unveiled expression changes associated with neurotransmission as well as motivation and substance use pathways. Sex differences were identified in nest building behavior, striatal neurotransmitter content, and ventral striatal gene expression.DiscussionIn summary, our study identified sex differences in specific behavioral, neurotransmitter, and gene expression phenotypes and gene set enrichment analysis identified significant enrichment of pathways associated with motivation and drug reward.

Adaptive group behavior of Fragile X mice in unfamiliar environments

In vitro and ex vivo studies have shown consistent indications of hyperexcitability in the Fragile X Messenger Ribonucleoprotein 1 (Fmr1) knockout mouse model of autism spectrum disorder. We recently introduced a method to quantify network-level functional excitation-inhibition ratio from the neuronal oscillations. Here, we used this measure to study whether the implicated synaptic excitation-inhibition disturbances translate to disturbances in network physiology in the Fragile X Messenger Ribonucleoprotein 1 (Fmr1) gene knockout model. Vigilance-state scoring was used to extract segments of inactive wakefulness as an equivalent behavioral condition to the human resting-state and, subsequently, we performed high-frequency resolution analysis of the functional excitation-inhibition biomarker, long-range temporal correlations, and spectral power. We corroborated earlier studies showing increased high-frequency power in Fragile X Messenger Ribonucleoprotein 1 (Fmr1) knockout mice. Long-range temporal correlations were higher in the gamma frequency ranges. Contrary to expectations, functional excitation-inhibition was lower in the knockout mice in high frequency ranges, suggesting more inhibition-dominated networks. Exposure to the Gamma-aminobutyric acid (GABA)-agonist clonazepam decreased the functional excitation-inhibition in both genotypes, confirming that increasing inhibitory tone results in a reduction of functional excitation-inhibition. In addition, clonazepam decreased electroencephalogram power and increased long-range temporal correlations in both genotypes. These findings show applicability of these new resting-state electroencephalogram biomarkers to animal for translational studies and allow investigation of the effects of lower-level disturbances in excitation-inhibition balance.

Neuronal hyperexcitability within developing cortical circuits is a common characteristic of several heritable neurodevelopmental disorders, including Fragile X Syndrome (FXS), intellectual disability and autism spectrum disorders (ASD). While this aberrant circuitry is typically studied from a neuron-centric perspective, glial cells secrete soluble factors that regulate both neurite extension and synaptogenesis during development. The nucleotide-mediated purinergic signalling system is particularly instrumental in facilitating these effects. We recently reported that within a FXS animal model, theFmr1 KO mouse, the purinergic signalling system is upregulated in cortical astrocytes leading to altered secretion of synaptogenic and plasticity-related proteins. In this study, we examined whether elevated astrocyte purinergic signalling also impacts neuronal morphology and connectivity of Fmr1 KO cortical neurons. Here, we found that conditioned media from primary Fmr1 KO astrocytes was sufficient to enhance neurite extension and complexity of both wildtype and Fmr1 KO neurons to a similar degree as UTP-mediated outgrowth. Significantly enhanced firing was also observed in Fmr1 KO neuron-astrocyte co-cultures grown on microelectrode arrays but was associated with large deficits in firing synchrony. The selective P2Y2 purinergic receptor antagonist AR-C 118925XX effectively normalized much of the aberrant Fmr1 KO activity, designating P2Y2 as a potential therapeutic target in FXS. These results not only demonstrate the importance of astrocyte soluble factors in the development of neural circuitry, but also show that P2Y purinergic receptors play a distinct role in pathological FXS neuronal activity.