Genetic reduction of MMP-9 in the Fmr1 KO mouse partially rescues prepulse inhibition of acoustic startle response

BackgroundIndividuals with Fragile X syndrome (FXS) and autism spectrum disorder (ASD) exhibit an array of symptoms, including sociability deficits, increased anxiety, hyperactivity, and sensory hyperexcitability. It is unclear how endocannabinoid (eCB) modulation can be targeted to alleviate neurophysiological abnormalities in FXS as behavioral research reveals benefits to inhibiting cannabinoid (CB) receptor activation and increasing endocannabinoid ligand levels. Here, we hypothesize that enhancement of 2-arachidonoyl-sn-glycerol (2-AG) in Fragile X mental retardation 1 gene knock-out (Fmr1 KO) mice may reduce cortical hyperexcitability and behavioral abnormalities observed in FXS.MethodsTo test whether an increase in 2-AG levels normalized cortical responses in a mouse model of FXS, animals were subjected to electroencephalography (EEG) recording and behavioral assessment following treatment with JZL-184, an irreversible inhibitor of monoacylglycerol lipase (MAGL). Assessment of 2-AG was performed using lipidomic analysis in conjunction with various doses and time points post-administration of JZL-184. Baseline electrocortical activity and evoked responses to sound stimuli were measured using a 30-channel multielectrode array (MEA) in adult male mice before, 4 h, and 1 day post-intraperitoneal injection of JZL-184 or vehicle. Behavior assessment was done using the open field and elevated plus maze 4 h post-treatment.ResultsLipidomic analysis showed that 8 mg/kg JZL-184 significantly increased the levels of 2-AG in the auditory cortex of both Fmr1 KO and WT mice 4 h post-treatment compared to vehicle controls. EEG recordings revealed a reduction in the abnormally enhanced baseline gamma-band power in Fmr1 KO mice and significantly improved evoked synchronization to auditory stimuli in the gamma-band range post-JZL-184 treatment. JZL-184 treatment also ameliorated anxiety-like and hyperactivity phenotypes in Fmr1 KO mice.ConclusionsOverall, these results indicate that increasing 2-AG levels may serve as a potential therapeutic approach to normalize cortical responses and improve behavioral outcomes in FXS and possibly other ASDs.

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.

Hypersensitivity to sounds is one of the prevalent symptoms in individuals with Fragile X syndrome (FXS). It manifests behaviorally early during development and is often used as a landmark for treatment efficacy. However, the physiological mechanisms and circuit-level alterations underlying this aberrant behavior remain poorly understood. Using the mouse model of FXS (Fmr1 KO), we demonstrate that functional maturation of auditory brainstem synapses is impaired in FXS. Fmr1 KO mice showed a greatly enhanced excitatory synaptic input strength in neurons of the lateral superior olive (LSO), a prominent auditory brainstem nucleus, which integrates ipsilateral excitation and contralateral inhibition to compute interaural level differences. Conversely, the glycinergic, inhibitory input properties remained unaffected. The enhanced excitation was the result of an increased number of cochlear nucleus fibers converging onto one LSO neuron, without changing individual synapse properties. Concomitantly, immunolabeling of excitatory ending markers revealed an increase in the immunolabeled area, supporting abnormally elevated excitatory input numbers. Intrinsic firing properties were only slightly enhanced. In line with the disturbed development of LSO circuitry, auditory processing was also affected in adult Fmr1 KO mice as shown with single-unit recordings of LSO neurons. These processing deficits manifested as an increase in firing rate, a broadening of the frequency response area, and a shift in the interaural level difference function of LSO neurons. Our results suggest that this aberrant synaptic development of auditory brainstem circuits might be a major underlying cause of the auditory processing deficits in FXS.SIGNIFICANCE STATEMENT Fragile X Syndrome (FXS) is the most common inheritable form of intellectual impairment, including autism. A core symptom of FXS is extreme sensitivity to loud sounds. This is one reason why individuals with FXS tend to avoid social interactions, contributing to their isolation. Here, a mouse model of FXS was used to investigate the auditory brainstem where basic sound information is first processed. Loss of the Fragile X mental retardation protein leads to excessive excitatory compared with inhibitory inputs in neurons extracting information about sound levels. Functionally, this elevated excitation results in increased firing rates, and abnormal coding of frequency and binaural sound localization cues. Imbalanced early-stage sound level processing could partially explain the auditory processing deficits in FXS.

Fragile X syndrome (FXS)—caused by FMR1 gene silencing—is a severe neurodevelopmental disorder characterized by intellectual disabilities that are often comorbid with seizures, sensory hypersensitivities, anxiety, social deficits, and repetitive behaviors. Neuronal hyperexcitability is an overarching neurophysiological characteristic of FXS that may underlie FXS symptoms. About 33% of Fmr1 KO mice from our colony exhibit spontaneous seizures, a newly observed phenotype that more closely parallels seizures in FXS, compared to the audiogenic seizure phenotype in Fmr1 KO mice. In addition, we and others show that Fmr1 KO mice, like individuals with FXS, have cortical EEG gamma‐band power alterations, and at the single‐cell level, have hyperexcitable pyramidal neurons in multiple brain regions. We are using a combinatorial approach—from behavioral to EEG to single‐cell experiments—to advance FXS drug discovery. Based on our observations of altered brain expression and in vivo function of serotonin 1A receptors (5‐HT1ARs) in Fmr1 KO mice and correction of the audiogenic seizure phenotype by our novel 2‐aminotetralin‐type 5‐HT1R modulator, FPT, we are testing the hypothesis that selectively activating 5‐HT1ARs prevents seizures and corrects neurophysiological abnormalities. We evaluated the efficacy of FPT (5.6 mg/kg), a potent and efficacious 5‐HT1AR agonist, to correct EEG abnormalities in Fmr1 KO mice. We also tested the antiepileptic effects of the selective 5‐HT1AR agonist, NLX‐112 (0.25‐2.5 mg/kg), and are currently testing the effects of FPT and NLX‐112 on CA1 pyramidal neuron hyperexcitability in Fmr1 KO mice. In parallel experiments, we are evaluating the pharmacology of FPT and NLX‐112 at each of the 5‐HT G protein‐coupled receptors. Recordings from above the left somatosensory cortex showed a significantly elevated high gamma (65‐100 Hz) power ratio in Fmr1 KO mice relative to control mice at baseline (n=16, P=0.0357) and after vehicle injection (n=16, P=0.0066), a genotype difference that FPT eliminated (n=15‐16, P=0.6279). Comparisons between baseline and first injection conditions also revealed an increased delta power in Fmr1 KO mice relative to controls. Separately, NLX‐112 prevented audiogenic seizures in Fmr1 KO mice (n=10‐12, P≤0.0002), and preliminary data suggest NLX‐112 and FPT modulate CA1 pyramidal neuron activity. For example, FPT (10 µM) showed a reversible reduction of firing frequency of hippocampal CA1 neurons in Fmr1 KO mice (n=8, P<0.05). Forthcoming experiments will include evaluating the effects of NLX‐112 on cortical EEG activity in Fmr1 KO and control mice and the effects of chronic NLX‐112 and FPT on spontaneous seizures in Fmr1 KO mice. Tests of the selective 5‐HT1AR antagonist, WAY100635, will be conducted to examine a 5‐HT1AR mechanism underlying positive outcomes of NLX‐112 and FPT. At present, our convergent data suggest that 5‐HT1AR activation may ameliorate neuronal hyperexcitability, at multiple levels of analysis, in Fmr1 KO mice. Potent and selective 5‐HT1AR agonists might be pharmacotherapeutic for FXS.

Fragile X syndrome (FXS) is the most common monogenetic cause of inherited intellectual disability and autism in humans. One of the well-characterized molecular phenotypes of Fmr1 KO mice, a model of FXS, is increased translation of synaptic proteins. Although this upregulation stabilizes in adulthood, abnormalities during the critical period of plasticity have long-term effects on circuit formation and synaptic properties. Using high-resolution quantitative proteomics of synaptoneurosomes isolated from the adult, developed brains of Fmr1 KO mice, we show a differential abundance of proteins regulating the postsynaptic receptor activity of glutamatergic synapses. We investigated the AMPA receptor composition and shuttling in adult Fmr1 KO and WT mice using a variety of complementary experimental strategies such as surface protein crosslinking, immunostaining of surface receptors, and electrophysiology. We discovered that the activity-dependent synaptic delivery of AMPARs is impaired in adult Fmr1 KO mice. Furthermore, we show that Fmr1 KO synaptic AMPARs contain more GluA2 subunits that can be interpreted as a switch in the synaptic AMPAR subtype toward an increased number of Ca2+-impermeable receptors in adult Fmr1 KO synapses.

Genetic reduction of muscarinic M 4 receptor modulates analgesic response and acoustic startle response in a mouse model of fragile X syndrome (FXS)

Long-lasting effects of minocycline on behavior in young but not adult Fragile X mice

Fragile X syndrome (FXS) is the most common form of inherited mental retardation and the most common known cause of autism. Loss of fragile X mental retardation protein (FMRP) in mice (Fmr1 KO) leads to altered synaptic and circuit maturation in the hippocampus that is correlated with alterations in hippocampal-dependent behaviors. Previous studies have demonstrated that loss of FMRP increased the rate of proliferation of progenitor cells and altered their fate specification in adult Fmr1 KO mice. While these studies clearly demonstrate a role for FMRP in adult neurogenesis in the hippocampus, it is not known whether the functional synaptic maturation and integration of adult-born dentate granule cells (abDGCs) into hippocampal circuits is affected in Fmr1 KO mice. Here, we used retroviral labeling to birthdate abDGCs in Fmr1 KO mice which allowed us to perform targeted patch clamp recording to measure the development of synaptic inputs to these neurons at precise time points after differentiation. The frequency and amplitude of spontaneous GABAergic events increased over the first three weeks after differentiation; however, this normal development of GABAergic synapses was not altered in Fmr1 KO mice. Furthermore, the relatively depolarized GABA reversal potential (EGABA) in immature abDGCs was unaffected by loss of FMRP as was the development of dendritic arbor of the adult generated neurons. These studies systematically characterized the functional development of abDGCs during the first four weeks after differentiation and demonstrate that the maturation of GABAergic synaptic inputs to these neurons is not grossly affected by the loss of FMRP.

Background:Fragile X syndrome (FXS) is the most common known inherited form of intellectual disability and the single genomic cause of autism spectrum disorders. It is caused by the absence of a fragile X mental retardation gene (Fmr1) product, FMRP, an RNA-binding translation suppressor. Elevated rates of protein synthesis in the brain and an imbalance between synaptic signaling via glutamate and γ-aminobutyric acid (GABA) are both considered important in the pathogenesis of FXS. In a mouse model of FXS (Fmr1 knockout [KO]), treatment with R-baclofen reversed some behavioral and biochemical phenotypes. A remaining crucial question is whether R-baclofen is also able to reverse increased brain protein synthesis rates.Methods:To answer this question, we measured regional rates of cerebral protein synthesis in vivo with the L-[1-14C]leucine method in vehicle- and R-baclofen–treated wildtype and Fmr1 KO mice. We further probed signaling pathways involved in the regulation of protein synthesis.Results:Acute R-baclofen administration corrected elevated protein synthesis and reduced deficits on a test of social behavior in adult Fmr1 KO mice. It also suppressed activity of the mammalian target of rapamycin pathway, particularly in synaptosome-enriched fractions, but it had no effect on extracellular-regulated kinase 1/2 activity. Ninety min after R-baclofen treatment, we observed an increase in metabotropic glutamate receptor 5 expression in the frontal cortex, a finding that may shed light on the tolerance observed in human studies with this drug.Conclusions:Our results suggest that treatment via activation of the GABA (GABA receptor subtype B) system warrants further study in patients with FXS.

The PDE10A Inhibitor TAK-063 Reverses Sound-Evoked EEG Abnormalities in a Mouse Model of Fragile X Syndrome.

Fragile X syndrome (FXS) is the most common form of inherited mental retardation. In exploring abnormalities associated with the syndrome, we have recently demonstrated abnormal vascular density in a FXS mouse model (Galvan and Galvez, ). One of the most prominent regulators of vascular growth is VEGF-A (Vascular Endothelial Growth Factor A), suggesting that FXS is associated with abnormal VEGF-A expression. In addition to its role in vascular regulation, VEGF-A also induces cellular changes such as increasing cell proliferation, and axonal and neurite outgrowth independent of its effects on vasculature. These VEGF-A induced cellular changes are consistent with FXS abnormalities such as increased axonal material, dendritic spine density, and cell proliferation. In support of these findings, the following study demonstrated that FXS mice exhibit increased expression of VEGF-A in brain. These studies suggest that increased VEGF-A expression in FXS is contributing to non-vascular FXS abnormalities. To explore the role of VEGF-A in mediating non-vascular FXS abnormalities, the monoclonal antibody Bevacizumab was used to block free VEGF-A. Bevacizumab treatment was found to decrease FXS Synapsin-1 expression, a presynaptic marker for synapse density, and reduce FXS testicle weight to control levels. Blocking VEGF-A also alleviated FXS abnormalities on novel object recognition, a test of cognitive performance. These findings demonstrate that VEGF-A is elevated in FXS brain and suggest that its expression promotes non-vascular FXS abnormalities. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 14-25, 2017.

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

Reduced perineuronal net expression in Fmr1 KO mice auditory cortex and amygdala is linked to impaired fear-associated memory.

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

Background:Fragile X syndrome (FXS) is the most common single gene inherited form of mental retardation, with behaviours at the extreme of the autistic spectrum. Subjects with FXS and fragile X...