Elsevier

Neuropharmacology

Volume 49, Issue 7, December 2005, Pages 1053-1066
Neuropharmacology

Suppression of two major Fragile X Syndrome mouse model phenotypes by the mGluR5 antagonist MPEP

https://doi.org/10.1016/j.neuropharm.2005.06.004Get rights and content

Abstract

Fragile X Syndrome is the most common form of inherited mental retardation worldwide. A Fragile X mouse model, fmr1tm1Cgr, 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 fmr1tm1Cgr 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 fmr1tm1Cgr mice. Genetic analysis revealed synergistic reactions between fmr1tm1Cgr and inbred AGS alleles. In addition, AGS sensitivity due to the fmr1tm1Cgr 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 fmr1tm1Cgr 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 fmr1tm1Cgr 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.

Introduction

Fragile X Syndrome (FXS) is an X-linked disorder most often resulting from expansion of a CGG trinucleotide repeat found in the 5′-untranslated region of the FMR1 gene. Once the expansion exceeds approximately 200 repeats, the FMR1 gene becomes hypermethylated and transcriptionally inactive, a condition that results in the loss of FMRP protein production in the affected cell. The primary symptom of FXS is mental retardation. In addition, autistic and hyperaroused behaviors, including hyperactivity, hypersensitivity to sensory stimuli, and EEG abnormalities are observed. More than 20% of those with FXS develop childhood seizures (Musumeci et al., 1999). Attention deficits and anxiety in novel situations are also common.

A mouse model of Fragile X Syndrome, with a disrupted Fmr1 allele designated fmr1tm1Cgr (“fmr1”), has been studied for the past decade (Bakker and Consortium, 1994). Biochemical analysis of the Fragile X (FX) mouse model has indicated that signaling through metabotropic glutamate receptors (mGluRs) may be altered. Changes in FMRP levels near synapses in response to mGluR group I signaling has been reported (Antar et al., 2004, Weiler et al., 1997). Of particular note for this study, it was recently reported that the CA1 region of fmr1tm1Cgr hippocampal slices exhibits increased long-term depression (LTD) when exposed to the mGluR group I receptor agonist DHPG (Huber et al., 2002).

LTD is an activity-dependent synaptic weakening which can be mediated at least in part by the group I mGluRs: mGluR1 and mGluR5. It had previously been shown using mGluR5 knockout mice that mGluR5 presence was required for mGluR1-mediated LTD (Huber et al., 2001); in contrast, LTD was reported to be normal in the CA1 region of mGluR1 knockout mice (Aiba et al., 1994). Furthermore, the mGluR5 agonist CHPG could induce LTD in the medial perforant pathway of the dentate gyrus (Camodeca et al., 1999), while the mGluR1 antagonist 4CPG alone did not block mGluR LTD (Oliet et al., 1997). Therefore, mGluR5 appeared to be essential for LTD in several assays.

Given the observation of altered LTD in fmr1 mice, FMRP might act downstream of mGluR group I receptors, in particular mGluR5, such that signaling is altered when FMRP levels are greatly reduced or lost. This raised the question, to be addressed in this study, whether in vivo evidence could be obtained for altered signaling through mGluR group I receptors in the fmr1tm1Cgr mouse.

MPEP (2-methyl-6-phenylethynyl pyridine hydrochloride) is a potent, specific, and blood brain barrier penetrable noncompetitive antagonist of mGluR5 receptors (Gasparini et al., 1999, Varney et al., 1999). MPEP suppresses clonic seizures induced by the mGluR5-selective agonist CHPG, and is also effective against sound-induced seizures in DBA/2 mice (Chapman et al., 2000). Therefore, MPEP was selected in this study for an initial assessment of the in vivo importance of mGluR5 receptor function in fmr1tm1Cgr mice. Two fmr1-specific and robust phenotypes were chosen for drug tests in FVB/NJ × C57BL/6 F1 hybrid mice: an elevated susceptibility to audiogenic seizures (AGS) and an increased tendency to move to the center of an open field (Yan et al., 2004).

It is shown here through the use of F1 hybrid mice that, in the absence of penetrant recessive modifier alleles, AGS sensitivity produced by the fmr1tm1Cgr allele is developmentally restricted, as is generally the case in human FXS. Furthermore, fmr1tm1Cgr mice have a more excitable audiogenic seizure pathway than wild-type littermates, and consequently, require more MPEP to achieve seizure suppression. Despite the complete acute effectiveness of MPEP in this AGS assay, fmr1tm1Cgr mice developed tolerance to MPEP's anticonvulsant activity over a period of days. In the open field test, MPEP was found to have an effect opposite to its previously described anxiolytic effect, yet the drug caused fmr1 mouse center field behavior to become statistically indistinguishable from that of wild-type. Therefore, modulation of mGluR5 signaling may allow amelioration of symptoms of Fragile X Syndrome. Some of the findings reported here have been previously published in abstract form (Yan et al., 2003).

Section snippets

Animals

Male and female inbred FVB/NJ (“FVB”) and C57Bl/6J (“C57”) mice, and F1 hybrids (“hybrid”) of the two strains, with or without an fmr1tm1Cgr allele (“fmr1” or “ko”), were used in the audiogenic seizure (AGS) testing. Data reported here are for the C57 male × FVB female cross; in several experiments the reverse cross was found to produce similar results. Male hybrids at one and three months of age were used for open field (OF) testing. The mice were housed in groups of five with a 12-h day–night

fmr1tm1Cgr AGS sensitivity is restricted during development

Male mice, 14–180 days old, were tested for sensitivity to audiogenic seizures (AGS) to determine the peak age of sensitivity. FVB fmr1tm1Cgr mice were sensitive to AGS induction over a wide range of ages, from 14 days postnatally to 94 days; by 150 days of age, no further seizures were observed (Fig. 1). C57 fmr1tm1Cgr mice also had a wide range of AGS sensitivity (from at least 15–47 days). By contrast, F1 hybrid (C57BL/6J × FVB/NJ) fmr1tm1Cgr mice did not show seizures at 15 days or after 31

Discussion

Several prior studies have suggested a link between mGluR group I signaling and FMRP: (1) FMRP levels were increased in wild-type synaptosomes and cultured cortical neurons after stimulation by the mGluR grI agonist DHPG (Todd et al., 2003a, Weiler et al., 1997), (2) MPEP substantially blocked a KCl-stimulated movement of FMRP and Fmr1 mRNA into dendrites of cultured hippocampal cells, while stimulation by DHPG led to a decrease in FMRP at synapses (Antar et al., 2004), and (3) increases in

Acknowledgements

We would like to thank Jeff and Debbie Stevenson for discussions and support, Ann M. Rogers for assistance with the seizure assays, and Jamie Feldman, Chris Sikorski, and Karina Illescas for assistance with the open field experiments and analysis. This work was supported by grants from the FRAXA Research Foundation.

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