Rescue of behavioral phenotype and neuronal protrusion morphology in Fmr1 KO mice

https://doi.org/10.1016/j.nbd.2008.04.002Get rights and content

Abstract

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.

Introduction

Fragile X syndrome (FXS) is the most common heritable form of mental retardation. The syndrome is caused by a lack of expression of FMRP (fragile X mental retardation protein), which is the protein product of the FMR1 gene. In most cases, the lack of expression is caused by expansion of a CGG repeat (> 200 U) in the 5ô UTR of the FMR1 gene, leading to methylation of both the CGG repeat and the promoter region, accompanied by transcriptional silencing. FMRP is an RNA-binding protein that associates with polyribosomes and is localized in neurons in the form of granules that move in a microtubule dependent manner with the speed of RNA transport (Antar et al., 2005, De Diego Otero et al., 2002, Wang et al., 2008). Moreover, FMRP has been shown to influence the translation efficacy of several of its target mRNAs (reviewed in Bagni and Greenough, 2005, Bardoni et al., 2006, Zalfa et al., 2007), which also implicates local translation at synaptic sites (Greenough et al., 2001, Muddashetty et al., 2007, Weiler et al., 1997, Weiler et al., 2004). In most cases, FMRP acts as a translational repressor (Laggerbauer et al., 2001, Lu et al., 2004). Therefore, FMRP is thought to be involved in the transport and/or the regulation of local mRNA translation at synaptic sites (Bagni and Greenough, 2005, Miyashiro et al., 2003, Weiler et al., 1997, Weiler et al., 2004). The presumed loss of translational regulation at synaptic sites might underlie the cognitive impairment in FXS (Huber et al., 2000).

Over the last few years, the metabotropic glutamate receptor (mGluR) theory of FXS has gained much support (Bear et al., 2004). The mGluR theory states that AMPA receptor internalization triggered by mGluR5 stimulation (Snyder et al., 2001), is exaggerated in Fmr1 KO mice, accounting for the enhanced hippocampal LTD found in knockout mice (Bear et al., 2004, Huber et al., 2002). Recently it was shown that FMRP deficient dendrites indeed show aberrant AMPA receptor trafficking resulting in a significantly reduced number of AMPA receptors at the plasma membrane (Nakamoto et al., 2007). Moreover, Fmr1 KO mice that are crossbred with mice that have a 50% reduction in mGluR5 expression were shown to be rescued in several phenotypic aspects (Dolen et al., 2007). It is hypothesized that FMRP normally is involved in the inhibition of the translation of several local mRNAs that are important for the mediation of AMPA receptor internalization. Since the amount of AMPA receptors in the postsynaptic density is correlated with protrusion shape, this might also explain the immature protrusion morphology that has been found in different brain areas of both fragile X patients and Fmr1 KO mice (Comery et al., 1997, Galvez and Greenough, 2005, Grossman et al., 2006, Hinton et al., 1991, Koekkoek et al., 2005, Nimchinsky et al., 2001). The mGluR theory has also boosted the search for therapeutic targets for FXS. An antagonist of mGluR5 receptors would theoretically counteract the increased amount of AMPA receptor internalization in Fmr1 KO neurons. Behavioral studies have shown that Fmr1 KO mice treated with the mGluR5 antagonist MPEP (2-methyl-6-(phenylethynyl)-pyridine hydrochloride) clearly display less sensitivity to audiogenic seizures and more wild type-like behavior in an open field test compared with untreated mice (Yan et al., 2005). Also in a Drosophila model based on loss of function of dfmr1, the single homolog of the FXR family of genes in the Drosophila genome, MPEP was able to rescue courtship behavior and mushroom body defects (McBride et al., 2005). However, the molecular mechanisms behind the effects of MPEP have not been elucidated.

In the present study, we show a defect in prepulse inhibition of acoustic startle (PPI) in Fmr1 KO mice compared to wild type littermates and a rescue of this behavioral phenotype by the mGluR5 antagonist MPEP. In addition, we demonstrate an altered protrusion morphology in Fmr1 KO primary hippocampal neurons that could be rescued using two different mGluR5 antagonists, MPEP and fenobam, rendering protrusion densities indistinguishable from wild type neurons.

Section snippets

Mouse models

Fmr1 KO mice were generated in our lab as described previously (Bakker et al., 1994, Mientjes et al., 2006). Both lines were used and were backcrossed to C57Bl/6J mice at least seven times. No differences were observed between both Fmr1 KO lines.

Prepulse inhibition of startle

Prepulse inhibition of startle (PPI) was measured by analysis of eye blink reactions of mice to acoustic stimuli, based on the magnetic distance measurement technique (MDMT) used for eye blink conditioning (Koekkoek et al., 2002, Koekkoek et al., 2005).

MPEP rescues prepulse inhibition of startle defect in Fmr1 KO mice

One of the most common clinical features of FXS is heightened sensitivity to sensory stimulation (Frankland et al., 2004, Miller et al., 1999). PPI is a widely used model to study basic sensorimotor processing and has shown to be related to mGluR signaling (Grauer and Marquis, 1999). In our mouse model, we examined PPI in wild type and Fmr1 KO mice. Mice were presented with a startling acoustic stimulus of 90 dB, which in the prepulse condition was preceded by a 70 dB pulse, 50 ms before the

Discussion

In this study we have shown a clear defect in PPI in Fmr1 KO mice measured by eye blink in response to loud sound. In support of the mGluR theory of FXS, this defect was rescued to wild type levels after treatment of the mice with 20 mg/kg of the mGluR5 antagonist MPEP. The impaired PPI response in Fmr1 KO mice is in line with sensorimotor gating deficits in FXS patients (Frankland et al., 2004). However, in the Frankland study, PPI was found to be increased rather than decreased in Fmr1 KO

Acknowledgments

We thank Dr. Casper C. Hoogenraad for helpful discussions and suggestions and Ronald Buijsen and Ingeborg Nieuwenhuizen for their technical assistance. This work was supported by the FRAXA Research Foundation (RW), NIH (NICHD R01 HD38038), (BAO and DLN), ZonMw 912-04-022 (BAO) and ZonMw 912-07-022 (RW).

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