6-methyl-2-(phenylethynyl)pyridine and Fragile-X-Syndrome

Disrupted metabotropic glutamate receptor 5 (mGluR5) signaling is implicated in many neuropsychiatric disorders, including autism spectrum disorder, found in fragile X syndrome (FXS). Here we report that intracellular calcium responses to the group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG) are augmented, and calcium-dependent mGluR5-mediated mechanisms alter the differentiation of neural progenitors in neurospheres derived from human induced pluripotent FXS stem cells and the brains of mouse model of FXS. Treatment with the mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) prevents an abnormal clustering of DHPG-responsive cells that are responsive to activation of ionotropic receptors in mouse FXS neurospheres. MPEP also corrects morphological defects of differentiated cells and enhanced migration of neuron-like cells in mouse FXS neurospheres. Unlike in mouse neurospheres, MPEP increases the differentiation of DHPG-responsive radial glial cells as well as the subpopulation of cells responsive to both DHPG and activation of ionotropic receptors in human neurospheres. However, MPEP normalizes the FXS-specific increase in the differentiation of cells responsive only to N-methyl-d-aspartate (NMDA) present in human neurospheres. Exposure to MPEP prevents the accumulation of intermediate basal progenitors in embryonic FXS mouse brain suggesting that rescue effects of GluR5 antagonist are progenitor type-dependent and species-specific differences of basal progenitors may modify effects of MPEP on the cortical development. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 419-437, 2017.

Topics: Animals; Cell Differentiation; Cerebral Cortex; Disease Models, Animal; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Female; Fragile X Syndrome; Humans; Male; Methoxyhydroxyphenylglycol; Mice; Mice, Inbred C57BL; Mice, Knockout; N-Methylaspartate; Neural Stem Cells; Pyridines; Receptor, Metabotropic Glutamate 5

The most common inherited monogenetic cause of intellectual disability is Fragile X syndrome (FXS). The clinical symptoms of FXS evolve with age during adulthood; however, neurophysiological data exploring this phenomenon are limited. The Fmr1 knockout (Fmr1KO) mouse models FXS, but studies in these mice of prefrontal cortex (PFC) function are underrepresented, and aging linked data are absent. We studied synaptic physiology and activity-dependent synaptic plasticity in the medial PFC of Fmr1KO mice from 2 to 12 months. In young adult Fmr1KO mice, NMDA receptor (NMDAR)-mediated long-term potentiation (LTP) is intact; however, in 12-month-old mice this LTP is impaired. In parallel, there was an increase in the AMPAR/NMDAR ratio and a concomitant decrease of synaptic NMDAR currents in 12-month-old Fmr1KO mice. We found that acute pharmacological blockade of mGlu5 receptor in 12-month-old Fmr1KO mice restored a normal AMPAR/NMDAR ratio and LTP. Taken together, the data reveal an age-dependent deficit in LTP in Fmr1KO mice, which may correlate to some of the complex age-related deficits in FXS.

Topics: Action Potentials; Animals; Disease Models, Animal; Electric Stimulation; Excitatory Postsynaptic Potentials; Fragile X Mental Retardation Protein; Fragile X Syndrome; Long-Term Potentiation; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neurons; Prefrontal Cortex; Pyridines; Receptor, Metabotropic Glutamate 5; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate

Fragile X syndrome (FXS) is the most common genetic cause for intellectual disability. Fmr1 knockout (KO) mice are an established model of FXS. Chronic pharmacological inhibition of metabotropic glutamate receptor 5 (mGlu5) in these mice corrects multiple molecular, physiological, and behavioral phenotypes related to patients' symptoms. To better understand the pathophysiology of FXS and the effect of treatment, brain activity was analyzed using functional magnetic resonance imaging in relation to learning and memory performance.. Wild-type (WT) and Fmr1 KO animals receiving chronic treatment with the mGlu5 inhibitor CTEP or vehicle were evaluated consecutively for 1) learning and memory performance in the inhibitory avoidance and extinction test, and 2) for the levels of brain activity using continuous arterial spin labeling based functional magnetic resonance imaging. Neural activity patterns were correlated with cognitive performance using a multivariate regression analysis. Furthermore, mGlu5 receptor expression in brains of untreated mice was analyzed by autoradiography and saturation analysis using [(3)H]-ABP688.. Chronic CTEP treatment corrected the learning deficit observed in Fmr1 KO mice in the inhibitory avoidance and extinction test and prevented memory extinction in WT and Fmr1 KO animals. Chronic CTEP treatment normalized perfusion in the amygdala and the lateral hypothalamus in Fmr1 KO mice and furthermore decreased perfusion in the hippocampus and increased perfusion in primary sensorimotor cortical areas. No significant differences in mGlu5 receptor expression levels between Fmr1 WT and KO mice were detected.. Chronic mGlu5 inhibition corrected the learning deficits and partially normalized the altered brain activity pattern in Fmr1 KO mice.

Topics: Animals; Avoidance Learning; Brain; Cognition; Disease Models, Animal; Electroshock; Excitatory Amino Acid Antagonists; Extinction, Psychological; Fragile X Mental Retardation Protein; Fragile X Syndrome; Imidazoles; Mice; Mice, Knockout; Oximes; Oxygen; Pyridines; Receptor, Metabotropic Glutamate 5; Tritium

Fragile X syndrome, the most common form of heritable mental retardation, is a developmental disorder with known effects within sensory systems. Altered developmental plasticity has been reported in the visual and somatosensory systems in Fmr1 knock-out (KO) mice. Behavioral studies have revealed maladaptive auditory responses in fragile X syndrome patients and Fmr1 KO mice, suggesting that adaptive plasticity may also be impaired in the auditory system. Here we show that, whereas tonotopic frequency representation develops normally in Fmr1 KO mice, developmental plasticity in primary auditory cortex is grossly impaired. This deficit can be rescued by pharmacological blockade of mGluR5 receptors. These results support the mGluR hypothesis of fragile X mental retardation and suggest that deficient developmental plasticity may contribute to maladaptive auditory processing in fragile X syndrome.

Topics: Animals; Auditory Cortex; Critical Period, Psychological; Disease Models, Animal; Excitatory Amino Acid Antagonists; Fragile X Mental Retardation Protein; Fragile X Syndrome; Mice; Mice, Knockout; Neuronal Plasticity; Neurons; Pyridines; Receptor, Metabotropic Glutamate 5

Fragile X syndrome (FXS) is a leading cause of intellectual disability. FXS is caused by loss of function of the FMR1 gene, and mice in which Fmr1 has been inactivated have been used extensively as a preclinical model for FXS. We investigated the behavioral pharmacology of drugs acting through dopaminergic, glutamatergic, and cholinergic systems in fragile X (Fmr1 (-/Y)) mice with intracranial self-stimulation (ICSS) and locomotor activity measurements. We also measured brain expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine biosynthesis. Fmr1 (-/Y) mice were more sensitive than wild type mice to the rewarding effects of cocaine, but less sensitive to its locomotor stimulating effects. Anhedonic but not motor depressant effects of the atypical neuroleptic, aripiprazole, were reduced in Fmr1 (-/Y) mice. The mGluR5-selective antagonist, 6-methyl-2-(phenylethynyl)pyridine (MPEP), was more rewarding and the preferential M1 antagonist, trihexyphenidyl, was less rewarding in Fmr1 (-/Y) than wild type mice. Motor stimulation by MPEP was unchanged, but stimulation by trihexyphenidyl was markedly increased, in Fmr1 (-/Y) mice. Numbers of midbrain TH+ neurons in the ventral tegmental area were unchanged, but were lower in the substantia nigra of Fmr1 (-/Y) mice, although no changes in TH levels were found in their forebrain targets. The data are discussed in the context of known changes in the synaptic physiology and pharmacology of limbic motor systems in the Fmr1 (-/Y) mouse model. Preclinical findings suggest that drugs acting through multiple neurotransmitter systems may be necessary to fully address abnormal behaviors in individuals with FXS.

Topics: Animals; Antipsychotic Agents; Aripiprazole; Behavior, Animal; Blotting, Western; Cocaine; Disease Models, Animal; Dopamine Uptake Inhibitors; Excitatory Amino Acid Antagonists; Fragile X Mental Retardation Protein; Fragile X Syndrome; Immunoenzyme Techniques; Male; Mice; Mice, Knockout; Motor Activity; Muscarinic Antagonists; Piperazines; Pyridines; Quinolones; Receptor, Metabotropic Glutamate 5; Reward; Trihexyphenidyl; Tyrosine 3-Monooxygenase

Pharmaceutical treatments are being developed to correct specific behavioural and morphological aspects of neurodevelopmental disorders such as mental retardation. Fragile X syndrome is an X-linked mental retardation with abnormal dendritic protrusions from neurons in the brain. Increased signalling via excitatory metabotropic glutamate receptor (mGluR) pathways is hypothesised to contribute to this disorder. Targeting these receptors has shown improvements in both behaviour and morphology with the Fmr1-KO mouse model for the syndrome. It is not known whether similar changes occur in excitatory synaptic activity following treatment with mGluR antagonists. We tested the effects of prolonged mGluR blockade on excitatory synaptic activity at three developmental time points in hippocampal slices. We observed a rescue effect of the antagonist MPEP upon spontaneous EPSC amplitude and charge at 2 weeks but not 1 week or 8-10 weeks of development. These data support the role of mGluR antagonist treatment for functional synaptic correction at an early developmental stage in a model for fragile X syndrome.

Topics: Animals; Animals, Newborn; Disease Models, Animal; Excitatory Amino Acid Antagonists; Female; Fragile X Mental Retardation Protein; Fragile X Syndrome; Hippocampus; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Organ Culture Techniques; Pyridines; Receptors, Metabotropic Glutamate; Synaptic Transmission

Abnormal dendritic spine morphology is a significant neuroanatomical defect in fragile X mental retardation. It has been suggested that overactive group 1 metabotropic glutamate receptor (mGlu) signaling is associated with the spine dysmorphology occurring in fragile X syndrome (FXS). Thus, group 1 mGlu became a new therapeutic target for the treatment of FXS.. The purpose of this study was to identify the effect of inhibition of mGlu signaling in FXS.. We observed the changes in dendritic spines after pharmacological modulation of mGlu signaling in an Fmr1 knockout (KO) mouse model.. The activation of group 1 mGlu resulted in elongation of dendritic spines in the cultured neurons derived from Fmr1 KO mice and wild-type (WT) mice. Antagonism of group 1 mGlu reduced the average spine length of Fmr1 KO neurons. Furthermore, systemic administration of the selective group 1 mGlu5 antagonist 2-methyl-6-phenylethynyl pyridine (MPEP) reduced the average spine length and density in the cortical neurons of Fmr1 KO mice at developmental age. For the adult mice, MPEP administration was less effective for the restoration of spine length. The percentage of immature spines showed a similar reduction in parallel to the changes of spine length. Temporary MPEP intervention with single-dose treatment did not show any effect.. These results show that MPEP administration could partially rescue the morphological deficits of dendritic spines in Fmr1 KO mice at developmental age.

Topics: Age Factors; Analysis of Variance; Animals; Animals, Newborn; Benzopyrans; Cells, Cultured; Dendritic Spines; Disease Models, Animal; Fragile X Mental Retardation Protein; Fragile X Syndrome; Hippocampus; Methoxyhydroxyphenylglycol; Mice; Mice, Inbred C57BL; Mice, Knockout; Neural Inhibition; Neurons; Pyridines; Receptors, Metabotropic Glutamate; Signal Transduction; Silver Staining

The most common cause of inherited mental retardation, fragile X syndrome, results from a triplet repeat expansion in the FMR1 gene and loss of the mRNA binding protein, fragile X mental retardation protein (FMRP). In the absence of FMRP, signaling through group I metabotropic glutamate receptors (mGluRs) is enhanced. We previously proposed a mechanism whereby the audiogenic seizures exhibited by FMR1 null mice result from an imbalance in excitatory mGluR and inhibitory GABA(B) receptor (GABA(B)R) signaling (Mol Pharmacol 76:18-24, 2009). Here, we tested the mGluR5-positive allosteric modulator 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB), the mGluR5 inverse agonist 2-methyl-6-(phenylethynyl)pyridine (MPEP), and GABA(B) receptor agonists, alone and in combination on receptor protein expression and audiogenic seizures in FMR1 mice. Single doses of MPEP (30 mg/kg), the GABA(B)R orthosteric agonist R-baclofen (1 mg/kg), or the GABA(B)R-positive allosteric modulator N,N'-dicyclopentyl-2-(methylthio)-5-nitro-4,6-pyrimidine diamine (GS-39783) (30 mg/kg), reduced the incidence of seizures. However, when administered subchronically (daily injections for 6 days), MPEP retained its anticonvulsant activity, whereas R-baclofen and GS-39783 did not. When administered at lower doses that had no effect when given alone, a single injection of MPEP plus R-baclofen also reduced seizures, but the effect was lost after subchronic administration. We were surprised to find that subchronic treatment with R-baclofen also induced tolerance to a single high dose of MPEP. These data demonstrate that tolerance develops rapidly to the antiseizure properties of R-baclofen alone and R-baclofen coadministered with MPEP, but not with MPEP alone. Our findings suggest that cross-talk between the G-protein signaling pathways of these receptors affects drug efficacy after repeated treatment.

Topics: Animals; Anticonvulsants; Baclofen; Benzamides; Blotting, Western; Cyclopentanes; Drug Interactions; Drug Tolerance; Epilepsy, Reflex; Excitatory Amino Acid Antagonists; Fragile X Mental Retardation Protein; Fragile X Syndrome; GABA Agonists; GABA Modulators; Mice; Mice, Inbred C57BL; Mice, Knockout; Pyrazoles; Pyridines; Pyrimidines; Receptors, GABA-B; Receptors, Kainic Acid; Receptors, Metabotropic Glutamate

Fragile X syndrome (FXS), a common inherited form of mental impairment and autism, is caused by transcriptional silencing of the fragile X mental retardation 1 (FMR1) gene. Earlier studies have identified a role for aberrant synaptic plasticity mediated by the metabotropic glutamate receptors (mGluRs) in FXS. However, many of these observations are derived primarily from studies in the hippocampus. The strong emotional symptoms of FXS, on the other hand, are likely to involve the amygdala. Unfortunately, little is known about how exactly FXS affects synaptic function in the amygdala. Here, using whole-cell recordings in brain slices from adult Fmr1 knockout mice, we find mGluR-dependent long-term potentiation to be impaired at thalamic inputs to principal neurons in the lateral amygdala. Consistent with this long-term potentiation deficit, surface expression of the AMPA receptor subunit, GluR1, is reduced in the lateral amygdala of knockout mice. In addition to these postsynaptic deficits, lower presynaptic release was manifested by a decrease in the frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs), increased paired-pulse ratio, and slower use-dependent block of NMDA receptor currents. Strikingly, pharmacological inactivation of mGluR5 with 2-methyl-6-phenylethynyl-pyridine (MPEP) fails to rescue either the deficit in long-term potentiation or surface GluR1. However, the same acute MPEP treatment reverses the decrease in mEPSC frequency, a finding of potential therapeutic relevance. Therefore, our results suggest that synaptic defects in the amygdala of knockout mice are still amenable to pharmacological interventions against mGluR5, albeit in a manner not envisioned in the original hippocampal framework.

Topics: Amygdala; Animals; Anti-Anxiety Agents; Autistic Disorder; Disease Models, Animal; Fragile X Mental Retardation Protein; Fragile X Syndrome; Male; Mice; Mice, Knockout; Neuronal Plasticity; Neurons; Pyridines; Receptors, AMPA; Receptors, Metabotropic Glutamate; Synapses

Fragile X syndrome (FXS) causes mental impairment and autism through transcriptional silencing of the Fmr1 gene, resulting in the loss of the RNA-binding protein fragile X mental retardation protein (FMRP). Cortical pyramidal neurons in affected individuals and Fmr1 knock-out (KO) mice have an increased density of dendritic spines. The mutant mice also show defects in synaptic and experience-dependent circuit plasticity, which are known to be mediated in part by dendritic spine dynamics. We used in vivo time-lapse imaging with two-photon microscopy through cranial windows in male and female neonatal mice to test the hypothesis that dynamics of dendritic protrusions are altered in KO mice during early postnatal development. We find that layer 2/3 neurons from wild-type mice exhibit a rapid decrease in dendritic spine dynamics during the first 2 postnatal weeks, as immature filopodia are replaced by mushroom spines. In contrast, KO mice show a developmental delay in the downregulation of spine turnover and in the transition from immature to mature spine subtypes. Blockade of metabotropic glutamate receptor (mGluR) signaling, which reverses some adult phenotypes of KO mice, accentuated this immature protrusion phenotype in KO mice. Thus, absence of FMRP delays spine stabilization and dysregulated mGluR signaling in FXS may partially normalize this early synaptic defect.

Topics: Animals; Animals, Newborn; Dendritic Spines; Disease Models, Animal; Excitatory Amino Acid Antagonists; Fragile X Mental Retardation Protein; Fragile X Syndrome; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Pseudopodia; Pyridines; Receptors, Metabotropic Glutamate

Fragile X syndrome (FXS) is caused by loss of the FMR1 gene product FMRP (fragile X mental retardation protein), a repressor of mRNA translation. According to the metabotropic glutamate receptor (mGluR) theory of FXS, excessive protein synthesis downstream of mGluR5 activation causes the synaptic pathophysiology that underlies multiple aspects of FXS. Here, we use an in vitro assay of protein synthesis in the hippocampus of male Fmr1 knock-out (KO) mice to explore the molecular mechanisms involved in this core biochemical phenotype under conditions where aberrant synaptic physiology has been observed. We find that elevated basal protein synthesis in Fmr1 KO mice is selectively reduced to wild-type levels by acute inhibition of mGluR5 or ERK1/2, but not by inhibition of mTOR (mammalian target of rapamycin). The mGluR5-ERK1/2 pathway is not constitutively overactive in the Fmr1 KO, however, suggesting that mRNA translation is hypersensitive to basal ERK1/2 activation in the absence of FMRP. We find that hypersensitivity to ERK1/2 pathway activation also contributes to audiogenic seizure susceptibility in the Fmr1 KO. These results suggest that the ERK1/2 pathway, and other neurotransmitter systems that stimulate protein synthesis via ERK1/2, represent additional therapeutic targets for FXS.

Topics: Animals; Disease Models, Animal; Fragile X Mental Retardation Protein; Fragile X Syndrome; Hippocampus; Isoenzymes; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Protein Biosynthesis; Pyridines; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Up-Regulation

Metabotropic glutamate receptor 5 (mGluR(5)) regulates the translation of amyloid precursor protein (APP) mRNA. Under resting conditions, mRNA is bound to and translationally repressed by the fragile X mental retardation protein (FMRP). Upon group 1 mGluR activation, FMRP dissociates from the mRNA and translation ensues. APP levels are elevated in the dendrites of primary neuronal cultures as well as in synaptoneurosomes (SN) prepared from embryonic and juvenile fmr-1 knockout (KO) mice, respectively. In order to study the effects of APP and its proteolytic product Abeta on Fragile X syndrome (FXS) phenotypes, we created a novel mouse model (FRAXAD) that over-expresses human APPSwe/Abeta in an fmr-1 KO background. Herein, we assess (1) human APP(Swe) and Abeta levels as a function of age in FRAXAD mice, and (2) seizure susceptibility to pentylenetetrazol (PTZ) after mGluR(5) blockade. PTZ-induced seizure severity is decreased in FRAXAD mice pre-treated with the mGluR(5) antagonist MPEP. These data suggest that Abeta contributes to seizure incidence and may be an appropriate therapeutic target to lessen seizure pathology in FXS, Alzheimer's disease (AD) and Down syndrome (DS) patients.

Topics: Age Factors; Amyloid beta-Protein Precursor; Animals; Anti-Anxiety Agents; Disease Models, Animal; Fragile X Mental Retardation Protein; Fragile X Syndrome; Gene Expression; Gene Silencing; Humans; Mice; Mice, Inbred C57BL; Mice, Knockout; Protease Nexins; Pyridines; Receptors, Cell Surface; RNA, Messenger; Seizures

Fragile X syndrome (FXS), a single gene disorder with an expanded CGG allele on the X chromosome, is the most common form of inherited cognitive impairment. The cognitive deficit ranges from mild learning disabilities to severe intellectual disability. The phenotype includes hyperactivity, short attention span, emotional problems including anxiety, social avoidance, poor eye contact, and hyperarousal to sensory stimuli. Imaging studies in FXS have clarified the impact of the FMR1 mutation on brain development and function by documenting structural abnormalities, predominantly in the caudate nucleus and cerebellum, and functional deficits in the caudate, frontal-striatal circuits, and the limbic system. On the basis of current research results, a targeted treatment for FXS will be available in the near future. Currently, a number of psychopharmacological agents are helpful in treating many of the problems in FXS including hyperactivity, attention deficits, anxiety, episodic aggression, and hyperarousal. Although the targeted treatments aim at strengthening synaptic connections, it is essential that these treatments are combined with learning programs that address the cognitive deficits in FXS.

Topics: Aggression; Alleles; Anxiety Disorders; Brain; Excitatory Amino Acid Antagonists; Female; Fragile X Mental Retardation Protein; Fragile X Syndrome; Humans; Intellectual Disability; Magnetic Resonance Imaging; Male; Mental Disorders; Phenotype; Pilot Projects; Point Mutation; Pyridines; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Social Behavior; Telomere

Fragile X syndrome is caused by the functional loss of the fragile X mental retardation 1 (FMR1) gene. Deletion of the FMR1 ortholog in Drosophila melanogaster (Fmr1) recapitulates many phenotypes associated with fragile X syndrome. We have discovered that Fmr1 mutant Drosophila die during development when reared on food containing increased levels of glutamate, which is consistent with the theory that FMR1 loss results in excess glutamate signaling. Using this lethal phenotype, we screened a chemical library of 2,000 compounds and identified nine molecules that rescued the lethality, including three that implicate the GABAergic inhibitory pathway. Indeed, GABA treatment rescued several known Fmr1 mutant phenotypes in flies, including mushroom bodies defects, excess Futsch translation and abnormal male courtship behavior. These data are consistent with GABAergic inhibition of the enhanced excitatory pathway in fragile X syndrome. In addition, our screen reveals that the muscarinic cholinergic receptors may have a role in fragile X syndrome in parallel to the GABAergic pathway. These results point to potential therapeutic approaches for treating fragile X syndrome.

Topics: Animals; Disease Models, Animal; Drosophila; Drosophila Proteins; Drug Evaluation, Preclinical; Female; Fragile X Mental Retardation Protein; Fragile X Syndrome; gamma-Aminobutyric Acid; Glutamic Acid; Male; Molecular Weight; Mutation; Phenotype; Pyridines; RNA, Messenger; Small Molecule Libraries

Lack of fragile X mental retardation protein (FMRP) causes Fragile X Syndrome, the most common form of inherited mental retardation. FMRP is an RNA-binding protein and is a component of messenger ribonucleoprotein complexes, associated with brain polyribosomes, including dendritic polysomes. FMRP is therefore thought to be involved in translational control of specific mRNAs at synaptic sites. In mice lacking FMRP, protein synthesis-dependent synaptic plasticity is altered and structural malformations of dendritic protrusions occur. One hypothesized cause of the disease mechanism is based on exaggerated group I mGluR receptor activation. In this study, we examined the effect of the mGluR5 antagonist MPEP on Fragile X related behavior in Fmr1 KO mice. Our results demonstrate a clear defect in prepulse inhibition of startle in Fmr1 KO mice, that could be rescued by MPEP. Moreover, we show for the first time a structural rescue of Fragile X related protrusion morphology with two independent mGluR5 antagonists.

Topics: Animals; Behavior, Animal; Cells, Cultured; Excitatory Amino Acid Antagonists; Fragile X Mental Retardation Protein; Fragile X Syndrome; Hippocampus; Imidazoles; Mice; Mice, Knockout; Microscopy, Confocal; Neurons; Phenotype; Pyridines; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Reflex, Startle

Fragile X Syndrome is a leading heritable cause of mental retardation that results from the loss of FMR1 gene function. Studies in mouse and Drosophila model organisms have been critical in understanding many aspects of the loss of function of the FMR1 gene in the human syndrome. Here, we establish that the zebrafish is a useful model organism for the study of the human fragile X syndrome and can be used to examine phenotypes that are difficult or inaccessible to observation in other model organisms. Using morpholino knockdown of the fmr1 gene, we observed abnormal axonal branching of Rohon-Beard and trigeminal ganglion neurons and guidance and defasciculation defects in the lateral longitudinal fasciculus. We demonstrate that this axonal branching defect can be rescued by treatment with MPEP [2-methyl-6-(phenylethynyl) pyridine]. This is consistent with an interaction between mGluR signalling and fmr1 function in neurite morphogenesis. We also describe novel findings of abnormalities in the abundance of trigeminal ganglion neurons and of craniofacial abnormalities apparently due to dysmorphic cartilage formation. These abnormalities may be related to a role for fmr1 in neural crest cell specification and possibly in migration.

Topics: Animals; Craniofacial Abnormalities; Disease Models, Animal; Excitatory Amino Acid Antagonists; Facial Bones; Fragile X Syndrome; Humans; Morphogenesis; Neural Crest; Neurites; Oligodeoxyribonucleotides, Antisense; Pyridines; Receptors, Metabotropic Glutamate; RNA-Binding Proteins; Zebrafish; Zebrafish Proteins

Fragile X Syndrome is the most common form of inherited mental retardation worldwide. A Fragile X mouse model, fmr1(tm1Cgr), with a disruption in the X-linked Fmr1 gene, has three substantial deficits observed in several strains: (1) sensitivity to audiogenic seizures (AGS), (2) tendency to spend significantly more time in the center of an open field, and (3) enlarged testes. Alterations in metabotropic glutamate receptor group I signaling were previously identified in the fmr1(tm1Cgr) mouse. In this study, we examined the effect of MPEP, an antagonist of the group I metabotropic glutamate receptor mGluR5, on audiogenic seizures and open field activity of fmr1(tm1Cgr) mice. Genetic analysis revealed synergistic reactions between fmr1(tm1Cgr) and inbred AGS alleles. In addition, AGS sensitivity due to the fmr1(tm1Cgr) allele was restricted during development. Examination of phenotypes combining mGluR5 inhibition and Fmr1 mutation indicated that absence of FMRP may affect mGluR5 signaling through indirect as well as direct pathways. All strains of fmr1(tm1Cgr) mice tested (FVB/NJ, C57BL/6J, and an F1 hybrid of the two) had a more excitable AGS pathway than wild-type, and consequently required more MPEP to achieve seizure suppression. At high doses of mGluR5 antagonists, a Fragile X specific tolerance (loss of drug activity) was observed. The tolerance effect could be overcome by a further increase in drug dose. In open field tests, MPEP reduced fmr1(tm1Cgr) center field behavior to one indistinguishable from wild-type. Therefore, mGluR5 antagonists were able to rescue two of the major phenotypes of the FX mouse. Modulation of mGluR5 signaling may allow amelioration of symptoms of Fragile X Syndrome.

Topics: Aging; Alleles; Animals; Drug Synergism; Epilepsy, Reflex; Exploratory Behavior; Female; Fragile X Mental Retardation Protein; Fragile X Syndrome; Genotype; Male; Mice; Mice, Inbred C57BL; Phenotype; Pyridines; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate