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mGlu5 as a potential therapeutic target for the treatment of fragile X syndrome

mGlu5 as a potential therapeutic target for the treatment of fragile X syndrome BioscienceHorizons Volume 6 • 2013 10.1093/biohorizons/hzt001 Review mGlu5 as a potential therapeutic target for the treatment of fragile X syndrome Lear Robertson Faculty of Biological Sciences, Institute of Membrane and Systems Biology, University of Leeds, Leeds LS2 9JT, UK. *Corresponding author: 1 Rochester Place, Charlbury, Chipping Norton, Oxfordshire OX7 3SF, UK. Email: lear.robertson@hotmail.co.uk Supervisor: Dr Steven Clapcote, Faculty of Biological Sciences, Institute of Membrane and Systems Biology, University of Leeds, Leeds LS2 9JT, UK. Fragile X syndrome (FXS) is the most common form of inherited mental retardation and the most common known cause of autism. It is caused by the expansion of a CGG trinucleotide repeat in the 5′ untranslated region of the fragile X mental retarda- tion 1 (FMR1) gene, which encodes the fragile X mental retardation protein (FMRP). FMRP negatively regulates group 1 (Grp 1) metabotropic glutamate receptor (mGlu a.k.a. mGluR) activity, and many FXS phenotypes are thought to be due to the over activity of the Grp 1 mGlu, mGlu5. This review evaluates the evidence for mGlu5 as a potential therapeutic target in the treat- ment of FXS. A 50% reduction in mGlu5 expression in Fmr1 knockout (KO) mice has been shown to reverse many FXS-relevant phenotypes including alterations in synaptic plasticity, increased dendritic spine density, increased basal hippocampal pro- tein synthesis, inhibitory avoidance extinction and susceptibility to audiogenic seizures. A negative modulator of mGlu5 may, therefore, be expected to have the same effect. In Fmr1 KO mice, Grp 1 mGlu antagonists, such as 2-methyl- 6-(phenylethynyl)pyridine (MPEP), fenobam and AFQ056, have been shown to reduce audiogenic seizures, reverse altered dendritic spine morphology, reduce excessive protein synthesis and improve behavioural abnormalities. MPEP, however, has failed to reverse altered long-term potentiation in the sensory neocortex or reduce machroorchidism. Clinical trials of mGlu5- negative modulators have had some positive outcomes but have had too few participants and were not performed over a long enough period to detect significant effects. Nevertheless, the prospects for development of mGlu5-negative modulators as FXS therapeutics are good and most research supports mGlu5 as a potential therapeutic target. Key words: fragile X syndrome, mGlu5, mGlu theory, Fmr1, FMRP Submitted on 6 September 2012; revised on 14 December 2012; accepted on 2 January 2013 Introduction et al., 1996). FMRP is a protein synthesis regulator at synapses, and functions as a translational repressor of target FXS is an inherited form of mental retardation first described mRNAs (Laggerbauer et al., 2001). It has also been hypoth- by Martin and Bell (1943) and was determined to be X-linked esized that FMRP plays a role in mRNA transportation along dominant with reduced penetrance after a large-scale segre- dendrites (Bassell and Warren, 2008). In FXS patients, there gation analysis by Sherman et al. (1984, 1985). Using scan- is a large decrease or complete silencing of the expression of ning electron microscopy, Harrison et al. (1983) found the the FMR1 gene, indicating that its loss of function is respon- X-chromosomal variant to be located at locus position sible for the syndrome. Xq27.3. Verkerk et al. (1991) became the first to clone the responsible gene: FMR1, which encodes the fragile X mental The causative mutation in FXS is a CGG expansion in the retardation protein (FMRP). FMRP is an RNA-binding pro- 5′ untranslated region of the FMR1 gene (Ashley et al., tein mainly present in the brain and testes and has been asso- 1993a). Normal individuals have a CGG trinucleotide repeat ciated with polyribosomes (Ashley et al., 1993b; Khandjian of between 6 and 54 (averaging 30 CGG units), while © The Author 2013. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Review Bioscience Horizons • Volume 6 2013 individuals with 55–200 have a fragile X premutation (Fu The mouse model et al., 1991). Individuals with >200 CGG repeats have the fragile X phenotype, females (XX) to a lesser extent due to The mouse Fmr1 gene is 97% homologous to the human compensation from the other, unaffected X chromosome. FMR1 gene (Ashley et al., 1993b). The first animal model of These CGG repeats lead to hypermethylation of the sur- FXS was created in 1994 by the Dutch Belgian fragile X rounding sequence including an upstream CpG island, caus- Consortium through the knock out (KO) of the Fmr1 gene in ing the silencing of FMR1 expression (Pieretti et al., 1991). mice (Bakker et al., 1994). FMRP was shown to be absent in FXS shows anticipation, which refers to the number of the the Fmr1 KO mice by western blotting and the phenotype trinucleotide repeats increasing from one generation to the appears consistent with, yet less severe than, human FXS; the next, meaning the risk of FXS increases in successive genera- mice had mild behavioural abnormalities, mild mental retar- tions (Fu et al., 1991). dation and machroorchidism. Most mouse models have been created in the same way: by KO of the Fmr1 gene and there- FXS is the most common inherited form of mental retar- fore do not include the expanded CGG trinucleotide repeat dation and is also responsible for 2–6% of autism cases, and so do not precisely model the causative mutation in FXS. making it the most common single gene mutation to cause The methylation state of the FMR1 promoter region depends autism (Reddy, 2005; Hagerman, Hoem and Hagerman, on the number of CGG repeats; Fmr1 KO mice only model 2010). Recent estimates of prevalence using DNA-based the fully methylated form of FXS, which represents only a diagnostic techniques revealed that FXS is present in ~1 in fraction of humans with FXS (Jacquemont et al., 2011). 4500 males and ~1 in 9000 females, with the premutation However, since KO of Fmr1 and the silencing of FMR1 by present in ~1 in 1000 males and ~1 in 400 females, it is pres- CGG expansion in human FXS both result in the lack of ent in every ethnic group (Crawford, Acuña and Sherman, expression of FMRP the mouse model is sufficiently similar 2001). to be useful in FXS research. In males, FXS is characterized by moderate-to-severe men- Mouse models of FXS do not replicate the anatomical tal retardation and autistic behaviours (Merenstein et al., abnormalities found in the brains of FXS patients. The size of 1996). There is also a developmental delay, and the majority the posterior vermis of the cerebellum in humans suffering of males suffer from attention deficit hyperactivity disorder from FXS is significantly reduced, but there is no difference in (ADHD) (Hagerman et al., 2009). Another neurological phe- the size of the posterior vermis in Fmr1 KO mice when com- notype present in 10–20% of boys with FXS and ~5% of pared with WT littermates (Kooy et al., 1999). However, girls is epilepsy, usually first occurring between the ages of 4 Fmr1 KO mice and humans with FXS both have more dense and 10 and ending later in childhood (Berry-Kravis, 2002; dendritic spines which tend to be ‘immature’ (long and thin) Incorpora et al., 2002). A connective tissue dysplasia results in appearance, but there is disagreement in the literature as to in the physical symptoms of FXS (narrow face, large ears, the precise phenotype (Irwin, Galvez and Greenough, 2000; machroorchidism and hyperextensible fingers) and it has Snyder et al., 2001; Vanderklish and Edelman, 2002). been hypothesized that hypothalamic dysfunction leads to the development of short bodies and limbs (Loesch et al., One reason that this mouse model of FXS has been 2003). In childhood, ~10% males with FXS develop Prader- accepted so readily is the broad range of behavioural pheno- Willi phenotype that consists of obesity and hyperphagia (de types that it reproduces (Bakker et al., 1994). FXS is a com- Vries et al., 1993; McLennan et al., 2011). plex disease with various neurological symptoms, many of which are expressed in the KO mice, including seizure sus- There is no cure for FXS, and the core symptom of mental ceptibility, social behaviour, sensitivity to stimuli and cogni- retardation is currently untreatable. However, some other tive performance (Musumeci et al., 2000; Dölen et al., 2007; symptoms of FXS may be improved pharmacologically or by de Vrij et al., 2008). Behavioural phenotype assays have thus behavioural interventions. α-adrenergic receptor agonists are far shown small effects in Fmr1 KO mice compared with used to treat the ADHD and selective serotonin reuptake humans with FXS. This may be because conventionally hip- inhibitors have been shown to be successful for treating anx- pocampus-dependent learning tasks, such as the Morris iety in over 50% of cases (Hagerman, Murphy and water maze, have been used, whereas a recent study indicates Wittenberger, 1988; Hagerman et al., 1994, 2002). A single that prefrontal cortex-dependent learning tasks could reveal anticonvulsant is often used to treat the epilepsy and antipsy- larger effects of Fmr1 KO (Krueger et al., 2011). chotics can help stabilize mood (Berry-Kravis and Potanos, 2004; Hagerman et al., 2009). There are no pharmaceuticals to treat the Prada-Willi phenotype, so the patient’s diet and The mGlu theory of FXS exercise must be regulated (Balko, 2005; Chen et al., 2007). Due to the lack of cure there is a clear need for new and bet- Mouse models have been instrumental in many break- ter therapies for FXS. This review evaluates the evidence for throughs in FXS research, revealing FXS as a disorder of metabotropic glutamate receptor 5 (mGlu5) as a potential long-term depression (LTD) and long-term potentiation therapeutic target in the treatment of FXS, and discusses how (LTP) in the cerebellum, the hippocampus and the prefrontal this might be achieved. cortex (Huber et al., 2002; Li et al., 2002; Koekkoek et al., 2 Bioscience Horizons • Volume 6 2013 Review article −/Y +/− 2005). The activation of both the N-methyl-d-aspartate reduction in mGlu5 [Fmr1 ; Grm5 ] (Lu et al., 1997). (NMDA) receptors and the Group (Grp) 1 mGlus results in Reducing mGlu5 expression by 50% in Fmr1 KO mice led to decreased postsynaptic 2-amino-3-(5-methyl-3-oxo-1,2-oxa- the rescue of various FXS-related phenotypes, including syn- zol-4-yl)propanoic acid (AMPA) and NMDA receptors due aptic plasticity alterations, increased dendritic spine density, to internalization (Oliet, Malenka and Nicoll, 1997). One increased basal hippocampal protein synthesis, exaggerated major mechanical dissimilarity between these two pathways inhibitor avoidance extinction and audiogenic seizure suscep- is that the mGlu-dependent LTD involves immediate mRNA tibility. This evidence supports the mGlu theory as well as the translation at the synapse, whereas NMDA-dependent LTD notion that pharmaceutical inhibition of mGlu5 could have a only requires mRNA translation if the LTD continues for similar effect. However, the 50% reduction in mGlu5 expres- more than a few hours (Huber, Kayser and Bear, 2000; sion in Fmr1 KO mice did not reverse the machroorchidism Manahan-Vaughan, Kulla and Frey, 2000). mGlu-dependent found in FXS (Dölen et al., 2007), indicating that mGlu5 does LTD is also irreversible and entails the loss of the synapse, not control testicle size. This raises the possibility that there unlike NMDA-dependent LTD which can be readily reversed may be other FXS phenotypes that are not rescued by reduced (Oliet, Malenka and Nicoll, 1997; Snyder et al., 2001). mGlu5 activity. Postnatal pharmacological inhibition of mGlu5 may not have the same effect as genetic inhibition of The study of FMRP in mGlu-dependent LTD was started mGlu5 in FXS patients and mouse models because sufficient when it was discovered that the activation of mGlus pro- damage may have been done already during prenatal develop- moted the synthesis of FMRP in synaptoneurosomes (Weiler ment to make the FXS phenotype irreversible. et al., 1997). mGlu-dependent LTD in Fmr1 KO mice was significantly increased compared with wild-type (WT) litter - mates but there was no difference in NMDA-dependent LTD, Pharmacological inhibition of mGlu5 indicating that the phenotype is exclusively linked to mGlu- dependent synaptic plasticity (Huber et al., 2002). mGlu5 is in mouse models of FXS the most prominent Grp 1 mGlu in the forebrain and thus it One behavioural phenotype that is present in both humans was hypothesized that mGlu5 activation stimulates the syn- with FXS and Fmr1 KO mice is heightened sensitivity to sen- thesis of proteins which enhance LTD, and FMRP inhibits sory stimuli. This has been shown to be related to mGlu sig- further synthesis of these proteins, therefore controlling the nalling and can be tested using prepulse inhibition (PPI), a LTD (Fig. 1A) (Bear, Huber and Warren, 2004). Excessive technique in which a reduced startle response is recorded synthesis of these proteins, as occurs in FXS, leads to inter- from a stimulus after a weaker prestimulus (Grauer and nalization of AMPA and NMDA receptors, causing the for- Marquis, 1999). de Vrij et al. (2008) studied PPI of acoustic mation of abnormally long and thin dendritic spines (Fig. 1B) startle responses in Fmr1 KO and WT mice. In WT mice, PPI (Irwin et al., 2000; Snyder et al., 2001; Vanderklish and was 73%, whereas it was 30% in Fmr1 KO mice, evidence of Edelman, 2002). the heightened sensitivity in the KO mice. Fmr1 KO mice treated with 20 mg/kg of the mGlu5 inhibitor 2-methyl-6- Mouse models and the mGlu theory (phenylethynyl)pyridine (MPEP) 30 mins before the experi- ment displayed PPI of 70%, an apparent rescue of the mGlu-dependent LTD was implicated in FXS by Huber et al. phenotype (de Vrij et al., 2008). However, Frankland et al. (2002). Paired-pulse facilitation and 3,5-dihydroxyphenylg- (2004) recorded increased PPI in Fmr1 KO mice compared lycine (DHPG) were independently used to induce LTD in with WT mice. This variation could be due to different exper- separate hippocampal slices of Fmr1 KO mice and WT lit- imental techniques, since de Vrij et al. (2008) measured the termates. The NMDA antagonist D-APV was used to prevent eyelid startle response, whereas Frankland et al. (2004) mea- NMDA-dependent LTD and so the experiment selectively sured the whole-body startle response. Moreover, while de recognized mGlu-dependent LTD. In the case of both the Vrij et al. (2008) found that MPEP increased PPI in WT mice, paired-pulse facilitation and DHPG, mGlu-dependent LTD this effect on PPI has not been observed in rats treated with was significantly enhanced in Fmr1 KO mice but not in WT MPEP (Henry et al., 2002; Zou et al., 2007). littermates, indicating that mGlu5 is overactive in FXS. MPEP has also been shown to reduce audiogenic seizures, This finding led to the hypothesis that the inhibition of which are characteristic of Fmr1 KO mice (Yan et al., 2005). mGlu5 may be a potential therapy for FXS (Bear, Huber and MPEP reduced audiogenic seizures in both Fmr1 KO and WT Warren, 2004). This was further investigated by Dölen et al. mice with three different genetic backgrounds (FVB/NJ, (2007), who generated Fmr1 KO mice with a 50% reduction C57BL/6J and an F1 hybrid of the two), although the dose in mGlu5 expression. This is possible because the genes for was larger in the KO mice. Application of MPEP also rescued FMRP and mGlu5 (FMR1 and GRM5) have single functional the open field phenotype, MPEP-treated Fmr1 KO mice spent homologues in mice (Fmr1 and Gmr5). By crossing Fmr1 KO as much time as WT mice in the centre of the open field mice with Gmr5 KO mice, offspring were produced with arena. These results support mGlu5 inhibition as a potential Fmr1 KO and 50% reduced mGlu5 expression. Complete KO therapy in FXS. Importantly, Yan et al. (2005) also discov- of mGlu5 causes impaired brain function and so it was impor- ered that mice can develop a tolerance to MPEP with repeat tant to study Gmr5 heterozygous mice with only a 50% 3 Review Bioscience Horizons • Volume 6 2013 Figure 1. Models representing the mGlu theory of FXS. (A) A model of the effect of mGlu5 activation and FMRP on mRNA translation at neuron synapses. mGlu5 activation causes AMPA receptor recycling and mRNA translation. This mRNA translation causes AMPA receptor internalization and is negatively regulated by FMRP. In FXS, FMRP is not present and so the mRNA translation is in excess as is the AMPA receptor internalization. (B) A model of the effect of FXS on dendritic spine shape. As AMPA and NMDA receptors are lost, the dendritic spines take on an ‘immature’ appearance (long and thin). Taken from Bear, Huber and Warren (2004). administration of the drug, which could prove to be a prob- case, MPEP may impair auditory acuity but would not be lem when developing mGlu5 antagonists as FXS treatments. targeting the core symptoms of FXS. Although the Yan et al. (2005) study appeared to show The de Vrij et al. (2008) study also determined the effect of that MPEP reverses two FXS-relevant phenotypes in Fmr1 mGlu5 antagonists MPEP and fenobam on dendritic spine KO mice, there could be another explanation for its effects. It morphology in Fmr1 KO mice. The morphology and density is conceivable that MPEP is reducing excitatory glutaminer- of dendritic spines has become a common test for the efficacy gic activity in the auditory pathway, thereby reducing the of pharmaceuticals in FXS. Although it is recognized that effect of the stimulus causing the seizures. If this were the there is some alteration in dendritic spine morphology in FXS 4 Bioscience Horizons • Volume 6 2013 Review article and Fmr1 KO mice, the literature is not in agreement on the mice, but in the KO mice the density of filopodia was signifi- specific alterations. It is generally accepted that the ratio of cantly greater. After treatment with the mGlu5 agonists ‘immature’ long thin spines (filopodia) to ‘mature’ mush- MPEP or fenobam, the ratio of normal spines to filopodia room shaped spines is greater in FXS and in Fmr1 KO mice matched those of the WT mice. The results of this study con- and that the density of the spines increased (Fig. 2) (Irwin, form to the predictions made by Dölen et al. (2007), who Galvez and Greenough, 2000). However, studies have pro- rescued the increased dendritic spine density by reducing duced results varying from reduced spine density in hippo- mGlu5 expression. campal cultures of Fmr1 KO mice (Braun and Segal, 2000) to The mGlu theory postulates that this altered spine mor- hugely dense filopodia (3–5 filopodia per 10 µm) (Antar phology is due to increased protein synthesis, which is regu- et al., 2006). These differing results could arise from varying lated by FMRP and stimulated by mGlu5 activation. FMRP definitions of ‘immature spines’ and the subjectivity behind binds with up to 4% of the brain’s mRNA in dendrites, their identification. To make the classification of these filopo- including its own, and suppresses its translation to protein dia more objective, de Vrij et al. (2008) considered all spines (Weiler et al., 1997; Laggerbauer et al., 2001). Translation of with length greater than the width to be filopodia and any FMRP therefore acts as a negative feedback mechanism to width greater than or equal to the length to be mature spines. stop excess translation caused by mGlu5 activation (Huber In this way, de Vrij et al. (2008) found that the density of et al., 2002) (Fig. 1A). Due to the lack of expression of FMRP mature spines was the same in both WT mice and Fmr1 KO in FXS, there is no negative feedback. The function of FMRP in mRNA translational regulation is incredibly complex and diverse, with much controversy surrounding its precise role. FMRP has been shown to be a translational activator as well as a repressor; it plays a part in translation initiation and elongation as well as being associated with the RNA-induced silencing complex. Many of the mRNAs that FMRP binds to, such as MAP1B, are factors in spine morphology (Gross, Berry-Kravis and Bassell, 2012). Aschrafi et al. (2005) observed decreased mRNA granules (shown to contain FMRP by western blot of rat brain lysate) in the brains of Fmr1 KO mice compared with WT. When treated with 35 mg/kg of MPEP, the size of the mRNA gran- ules were significantly increased in both Fmr1 KO and WT mice. This suggests that granules are stabilized by FMRP and destabilized by mGlu5 activity. This study does not directly measure protein synthesis; the link between mRNA granules and level of protein synthesis is only hypothetical. It is unclear why Aschrafi et al. (2005) performed a western blot of brain lysate from rats to prove the presence of FMRP in mRNA granules: the study does not show that FMRP is present in mRNA granules in mice brains. This increased protein synthesis leads to altered synaptic plasticity. In Fmr1 KO mice, the most characterized form of altered synaptic plasticity is enhanced mGlu-dependent LTD in the Shaffer collaterals of the CA1 region of the hippocam- pus (Huber, Roder and Bear, 2001; Huber et al., 2002). To date, no studies have investigated the effects of mGlu5-nega- tive modulation on LTD anywhere in the brain. However, the effect of the Grp 2 mGlu antagonist LY341495 on mGlu- dependent LTD in Fmr1 KO mice has been researched in CA1 neurons (Choi et al., 2011), the rationale being that antago- nism of DmGluA, the Drosophila flies sole mGlu homologue, has been shown to rescue memory and social interaction (McBride et al., 2005). DmGluA has both Grp 1 and 2 mGlu activity. Chronic treatment with the Grp 2 mGlu antagonist Figure 2. Spine morphologies of Golgi-impregnated neurons. (A) LY341495 for 8 weeks rescued DHPG-induced LTD in adult Dendritic spines of human affected with FXS. (B) Dendritic spines of a Fmr1 KO mice, but increased DHPG-induced LTD in WT human without FXS. The density of long thin spines is much greater in mice. The reason for the contrasting effects in Fmr1 KO and (A) than in (B). Taken from Irwin, Galvez and Greenough, (2000). 5 Review Bioscience Horizons • Volume 6 2013 WT mice is unknown but Choi et al. (2011) hypothesized that function that mGlu5-negative modulators would rescue may it could be due to some sort of compensation in the system. be the regulation of mGlu5 activity. However, LY341495 is not completely selective for Grp 2 MPEP is not a specific inhibitor of mGlu5, and can inhibit mGlus, as it is also a Grp 3 and a Grp 1 mGlu antagonist. NMDA receptors when at high concentrations (Lea, Movsesyan LY341495 has a 1000-fold greater affinity for Grp 2 mGlus and Faden, 2005), thus raising the possibility that MPEP than Grp 1, and 10-fold greater for Grp 2 than for Grp 3 treatment could potentially have adverse effects. Most stud- (Kingston et al., 1998). The dose was also kept at the lowest ies have tried to use MPEP at concentrations low enough not level previously shown to reverse the in vivo effects of a Grp 2 to affect NMDA activity, but it is conceivable that some con- mGlu agonist (Johnson et al., 1999). Nevertheless, it cannot clusions drawn from research involving MPEP could be due be ruled out that the antagonistic effect on the Grp 1 or Grp 3 to its NMDA-inhibiting action rather than its ability to mGlus did not cause the phenotype rescue. inhibit mGlu5. MPEP cannot be used as a therapy in humans In contrast to LDP, LTP is reduced in the sensory neocor- with FXS owing to its toxicity. It also has a very short half-life tex of Fmr1 KO mice but not in the hippocampus (Fig. 3) (~1 h in C57BL/6J mice, Anderson et al., 2003) meaning that (Li et al., 2002). Wilson and Cox (2007) showed that in the administration of the drug would have be more frequent vitro LTP in the visual neocortex in WT mice is not only than is practicable. NMDA-dependent but primarily due to mGlu activity. They AFQ056 is a drug developed by Novartis which could proposed that the reduced LTP measured in the visual cortex potentially be used to treat FXS in humans since it does not of Fmr1 KO mice is due to reduced mGlu5 activity. When have the same drawbacks as MPEP, such as toxicity and the mGlu5 inhibitor MPEP was applied to the visual cortex short half-life. Levenga et al. (2011) tested the effect of of Fmr1 KO mice, there was no significant difference in LTP AFQ056 on the spine morphology and PPI of Fmr1 KO between MPEP-treated and control Fmr1 KO mice. This mice. AFQ056 was shown to restore PPI in Fmr1 KO mice suggests that mGlu5-mediated synaptic plasticity is in deficit from 22 to 48% and also increased PPI in WT mice. AFQ056 in the visual cortex, in contrast to the hippocampus where also affected spine morphology: when administered to Fmr1 mGlu5-mediated synaptic plasticity is in excess. If this is the KO mice in three different concentrations (10 nm, 1 µ M and case, it poses serious problems for mGlu5 as a potential ther- 10 µ M), the hippocampal dendritic spine length decreased. apeutic target; since it shows that the relationship between Interestingly, AFQ056 increased spine density and decreased FMRP and mGlu5 is more complicated than originally spine width. This is in contrast to the results of the in vivo thought. It also implies that FMRP-regulated events may be study by Dölen et al. (2007), in which a decrease in the spine specific to particular brain areas, rendering mGlu5 inhibi- density and an increase in the spine width was observed tion useless or perhaps even harmful in some areas of the when mGlu5 expression was reduced by 50% in Fmr1 KO brain. mice. As a relatively new drug, very little research has been Yan et al. (2005) postulated that mGlu5-negative modula- conducted using AFQ056. There have not yet been any stud- tors, such as MPEP, will only have an effect on phenotypes ies published that confirm AFQ056 as a selective mGlu5 caused by FMRP-independent signalling and may have no antagonist as it is described by Levenga et al. (2011). Much effect on FMRP-dependent signalling, thus, some FXS phe- more research is needed before it can be considered a poten- notypes will remain (Fig. 4). Essentially, the only FMRP tial therapy. Figure 3. Graph showing the LTP response to tetanic stimulation in WT and in Fmr1 KO mice. Taken from deep layers of the visual neocortex in the presence of low concentration of the GABA antagonist bicuculline. LTP is significantly reduced in the Fmr1 KO mice compared with the WT mice over 60 min of tetanic stimulation. MPEP did not rescue the reduced LTP in Fmr1 KO mice (data not shown). Taken from Wilson and Cox (2007); Copyright 2007 National Academy of Sciences, USA. 6 Bioscience Horizons • Volume 6 2013 Review article conducted on 30 individuals with FXS (Jacquemont et al., 2011). Individuals were screened to determine the extent of the methylation of their FMR1 promoter. Interestingly, only individuals with a fully methylated promoter and no FMR1 mRNA detected in the blood showed significant improve- ments in comparison with the control group when measured using various behavioural rating scales. This suggests that screening for full methylation could determine which patients would benefit from mGlu5 antagonists. The response to treatment in patients with partial methyla- tion of the FMR1 promoter was varied. Jacquemont et al. (2011) propose that this variation in response is due to differ- ence in methylation states and therefore to different degrees of mGlu5 hyperactivity (Fig. 5). The baseline scores of the behav- ioural rating scales indicate a more severe phenotype in the fully methylated participants which supports this theory; however, this difference is not statistically significant. The lack of correla- tion between FMR1 mRNA in the blood and response to AFQ056 casts doubt over this hypothesis, although it could be explained by variance in tissue-specific methylation or variance in the translation of FMR1 mRNA to FMRP. An assessment of Figure 4. A diagram of MPEP effect on mGlu5 signalling. mGlu5 signalling: FMRP independent (A), and FMRP dependent (B). Negative FMRP levels may have helped draw conclusions as to why modulators of mGlu5 may only have effect on A and not on B. This is there was so much variation in response amongst the partially because by negatively modulating mGlu5, FMRP’s inhibitory effect on methylated subpopulation but no such analysis was carried mGlu5 signalling (A) will be mimicked, whereas the FMRP itself will still out. The small number of trial patients (seven with fully meth- not be present and therefore no other functions of FMRP (B) will be ylated FMR1 promoters) makes it difficult to draw any solid rescued. conclusions from this study. Future trials with more partici- pants may offer clues to the mechanism behind why fully meth- Clinical trials with mGlu5-negative ylated participants responded more consistently to treatment. modulators in FXS Jacquemont et al. (2011) chose to use behavioural response to measure the efficacy of AFQ056. Measurement of change In recent years, the positive results of studies into mGlu5- negative modulators in preclinical animal models has led to the development of pharmaceuticals and clinical trials in humans with FXS that target mGlu5, including fenobam, AFQ056 (Novartis), STX107 (Seaside Therapeutics) and RG7090 (Hoffmann-LaRoche). A pilot open label, single-dose trial of fenobam was con- ducted on 12 subjects (6 male, 6 female) with FXS (Berry- Kravis et al., 2009). The main aim of the experiment was to determine the safety of the drug and identify any significant adverse effects, of which there was none. Each patient received a single dose of fenobam (between 50 and 150 mg) and was then screened for vital signs and side effects during the following 6 h. 50% of the patients had amended PPI after drug administration ranging from 23.7 to 44.2% improve- ment. It is known that the placebo effect is exaggerated in individuals with mental retardation (Sandler, 2005) and so Figure 5. Methylation state of the FMR1 promoter affects the efficacy the open label nature of this trial may have some influence on of AFQ056. Schematic showing the influence that the methylation the results. The results also suffered due to the limited num- state of the FMR1 promoter has over the efficacy of AFQ056 in FXS ber of patients in the trial and the fact that they were only patients. Participants with fully methylated promoters (only given a single dose. However, the lack of adverse effects is methylated DNA was detected) experienced a behavioural promising for future trials. improvement in response to AFQ056, whereas participants with partially methylated promoters (methylated and non-methylated DNA A double-blind, two-treatment, two-period crossover trial was detected) had varying responses and no statistically significant of the subtype-selective mGlu5 inhibitor AFQ056 was improvement in behaviour. Taken from Pouladi (2011). 7 Review Bioscience Horizons • Volume 6 2013 in PPI or eye tracking would have been a more objective way human disease. In the mouse models, the behavioural pheno- to quantify the response. Also, to see a developmental type is less obvious and the symptoms much milder than in improvement in the participants rather than just symptomatic human FXS. Overall, the prospect for the development of improvements, future trials will have to be conducted over mGlu5-negative modulators as a therapy appears good, as is significantly longer time periods, particularly in older patients. evident from the clinical trials now underway. Currently, there are other mGlu5-negative modulators One important question in need of answering is whether or such as STX107 and RG7090, which are undergoing clinical not the effect of FXS during prenatal development is reversible trials but the result have yet to be released. by pharmaceuticals. The application of mGlu5-negative mod- ulators is unlikely to be a practical option as the average age of diagnosis is at ~35–37 months in males and later in females Discussion (Bailey et al., 2009). In most models, it has been shown that at least some of the phenotypes are reversed by treatment with Much of the evidence in preclinical animal models of FXS mGlu5-negative modulators in adulthood. The clinical trials indicates that mGlu5-negative modulators may eventually be during prenatal development that have been performed so far used therapeutically. The studies using mouse models are tell us very little about the long-term effects of mGlu5-negative summarized in Table 1. There are minor inconsistencies modulators. Future trials need to be carried out over a much within these studies, many of which could be due to differing longer time period and with many more participants to find experimental techniques. Taken collectively, however, these out to what extent these drugs can be beneficial. works tend to support the mGlu theory put forward by Bear, Huber and Warren (2004) but do not paint a complete pic- Twenty-four of the 30 participants in the Jacquemont ture. Some results indicate that Grp 2 mGlus may also be et al. (2011) study experienced adverse effects, mostly fatigue implicated in the mGlu theory (Wilson and Cox, 2007; Choi and headaches but some suffered from hyperlipasemia, et al., 2011). The major limitation within the preclinical hyperamylasemia, increased hepatic enzymes and increased studies is the fundamental problem of modelling human blood creatinine phosphokinase. At higher doses these diseases in animals. The validity of the models depends on adverse effects may become more severe and as this was a the extent to which the animal disease is analogous to the relatively small trial there may be other more dangerous side Table 1. Summary of studies of mGlu5-negative modulation in Fmr1 KO mice Therapy Reversed phenotype Target Study Comments/discrepancies Genetic rescue; Altered synaptic plasticity, mGlu5 Dölen et al. 50% reduction in the mGlu5 expression was unable to 50% reduction dendritic spine density, basal (2007) rescue machroorchidism in mGlu5 hippocampal protein synthesis, expression inhibitor avoidance extinction, audiogenic seizures MPEP Decreased PPI of acoustic startle Grp 1 mGlus de Vrij et al. Inconsistent with the findings of Frankland et al. (2004) response (2008) who observed increased PPI in Fmr1 KO mice compared with WT. Hugely varying alterations in spine morphol- ogy was observed in Fmr1 KO mice (Braun and Segal, 2000; Antar et al., 2006) MPEP, fenobam Increased ratio of filopodia to Grp 1 mGlus de Vrij et al. As above. mature dendritic spines (2008) MPEP Audiogenic seizures, centre field Grp 1 mGlus Yan et al. Reduction in auditory seizures could be due to reduced behaviour (2005) glutaminergic activity in the auditory pathway decreasing the effect of the stimulus. MPEP was also unable to rescue increased size of testis MPEP Excessive protein synthesis Grp 1 mGlus Aschrafi No evidence that FMRP is present in mRNA granules in et al. (2004) mouse brains. Link between the mRNA granule size and the level of protein synthesis is only hypothetical LY341495 Increased DHPG-induced LTD in Grp 2 mGlus Choi et al. LY341495 has a very small antagonistic effect on Grps the CA1 region of the (2011) 1 and 3 mGlus hippocampus MPEP None Gp 1 mGlus in Wilson and MPEP did not rescue excess LTP in the sensory sensory neocortex Cox (2007) neocortex AFQ056 Decreased PPI, increased spine Grp 1 mGlus Levenga AFQ056 increased spine density and decreased spine length et al. (2011) width which is in contrast to Dölen et al. (2007) in which the decreased mGlu5 expression did the opposite 8 Bioscience Horizons • Volume 6 2013 Review article Bakker, C. E., Verheij, C., Willemsen, R. et al. (1994) Fmr1 knockout mice: effects that did not present themselves. The results of the a model to study fragile X mental retardation, The Dutch-Belgian Wilson and Cox (2007) study indicate a variance in the Fragile X Consortium, Cell, 78, 23–33. mGlu5 activity across different brain regions; this is a poten- tial problem with regard to the therapeutic use of mGlu5- Balko, K. (2005) Red Yellow Green System for Weight Management. negative modulators. Specifically mGlu5 inhibition has been Toronto: Ontario Prader-Willi Syndrome Association. shown to have no effect on the increased LTP in the visual cortex and may have a negative effect by decreasing visual Bassell, G. J. and Warren, S. T. (2008) Fragile X Syndrome: loss of local cortex LTD (Wilson and Cox, 2007). mRNA regulation alters synaptic development and function, Neuron, 60, 201–214. Inhibition of mGlu5 does not offer a complete cure for FXS, since some symptoms such as machroorchidism have Bear, M. F., Huber, K. M. and Warren, S. T. (2004) The mGluR theory proved to be resistant to mGlu5-negative modulation and of  fragile X mental retardation, Trends in Neuroscience, 27, there may be other refractory symptoms yet to be identified 370–377. (Dölen et al., 2007). Perhaps inhibition of mGlu5 will help to Berry-Kravis, E. (2002) Epilepsy in fragile X syndrome, Developmental control many of the symptoms and, along with other drugs, Medicine and Child Neurology, 44, 724–728. significantly improve the quality of life of the patient and the carers. With further research into mGlu5-negative modula- Berry-Kravis, E. and Potanos, K. (2004) Psychopharmacology in fragile X tors in animal models and through clinical trials, their full syndrome—present and future, Mental Retardation and therapeutic potential will be revealed. Developmental Disabililities Research Reviews, 10, 42–48. Berry-Kravis, E., Hessl, D., Coffey, S. et al. (2009) A pilot open label, single Acknowledgments dose trial of fenobam in adults with fragile X syndrome, Journal of Medical Genetics, 46, 266–271. Thanks to Steve Clapcote for his help and advice, to Peter Wright for editing and to Francesca Deane for motivation. Braun, K. and Segal, M. (2000) FMRP involvement in formation of syn- apses among cultured hippocampal neurons, Cerebral Cortex, 10, 1045–1052. Author biography Chen, C., Visootsak, J., Dills, S. et  al. (2007) Prader-Willi syndrome: an I graduated from the University of Leeds in July 2012 with a update and review for the primary pediatrician, Clinical Pediatrcs, degree in neuroscience. My particular interests are neurologi- 46, 580–591. cal disorders and the development of therapies for them. I am currently applying for postgraduate study. Choi, C. H., Schoenfeld, B. P., Bell, A. J. et  al. (2011) Pharmacological reversal of synaptic plasticity deficits in the mouse model of fragile X syndrome by group II mGluR antagonist or lithium treatment, References Brain Research, 1380, 106–119. Anderson, J. J., Bradbury, M. J., Giracello, D. R. et al. (2003) In vivo recep- Crawford, D. C., Acuña, J. M. and Sherman, S. L. (2001) FMR1 and the tor occupancy of mGlu5 receptor antagonists using the novel radio- fragile X syndrome: human genome epidemiology review, Genetics ligand [3H]3-methoxy-5-(pyridin-2-ylethynyl)pyridine), European in Medicine, 3, 359–371. Journal of Pharmacology, 473, 35–40. de Vries, B. B., Fryns, J. P., Butler, M. G. et al. (1993) Clinical and molecular Antar, L. N., Li, C., Zhang, H. et al. (2006) Local functions for FMRP in axon studies in fragile X patients with a Prader-Willi-like phenotype, growth cone motility and activity-dependent regulation of filopodia Journal of Medical Genetics, 30, 761–766. and spine synapses, Molecular and Cellular Neuroscience, 32, 37–48. de Vrij, F. M. S., Levenga, J., van der Linde, H. C. et  al. (2008) Rescue of Aschrafi, A., Cunningham, B. A., Edelman, G. M. et al. (2005) The fragile X behavioral phenotype and neuronal protrusion morphology in mental retardation protein and group I metabotropic glutamate Fmr1 KO mice, Neurobiology of Disease, 31, 127–132. receptors regulate levels of mRNA granules in brain, Proceedings of the National Academy of Sciences of the USA, 102, 2180–2185. Dölen, G., Osterweil, E., Shankaranarayana Rao, B. S. et  al. (2007) Correction of fragile X syndrome in mice, Neuron, 56, 955–962. Ashley, C. T., Wilkinson, K. D., Reines, D. et al. (1993a) FMR1 protein: con- served RNP family domains and selective RNA binding, Science, 262, Frankland, P. W., Wang, Y., Rosner, B. et  al. (2004) Sensorimotor gating 563–566. abnormalities in young males with fragile X syndrome and Fmr1- knockout mice, Molecular Psychiatry, 9, 417–425. Ashley, C. T., Sutcliffe, J. S., Kunst, C. B. et al. (1993b) Human and murine FMR-1: alternative splicing and translational initiation downstream Fu, Y. H., Kuhl, D. P., Pizzuti, A. et al. (1991) Variation of the CGG repeat at of the CGG-repeat, Nature Genetics, 4, 244–251. the fragile X site results in genetic instability: resolution of the Sherman paradox, Cell, 67, 1047–1058. Bailey, D. B., Raspa, M., Bishop, E. et  al. (2009) No change in the age of diagnosis for fragile X syndrome: findings from a National Parent Grauer, S. M. and Marquis, K. L. (1999) Intracerebral administration of Survey, Pediatrics, 124, 527–533. metabotropic glutamate receptor agonists disrupts prepulse 9 Review Bioscience Horizons • Volume 6 2013 inhibition of acoustic startle in Sprague-Dawley rats, Psycho- receptor subtype expressing cells, Neuropharmacology, 38, 1519– pharmacology (Berl.), 141, 405–412. 1529. Gross, C., Berry-Kravis, E. M. and Bassell, G. J. (2012) Therapeutic strate- Khandjian, E. W., Corbin, F., Woerly, S. et  al. (1996) The fragile X mental gies in fragile X syndrome: dysregulated mGluR signaling and retardation protein is associated with ribosomes, Nature Genetics, beyond, Neuropsychopharmacology, 37, 178–195. 12, 91–93. Hagerman, R. J., Murphy, M. A. and Wittenberger, M. D. (1988) A con- Kingston, A. E., Ornstein, P. L., Wright, R. A. et  al. (1998) LY341495 is a trolled trial of stimulant medication in children with the fragile X nanomolar potent and selective antagonist of group II metabo- syndrome, American Journal of Medical Genetics, 30, 377–392. tropic glutamate receptors, Neuropharmacology, 37, 1–12. Hagerman, R. J., Fulton, M. J., Leaman, A. et al. (1994) A survey of fluox - Koekkoek, S. K. E., Yamaguchi, K., Milojkovic, B. A. et al. (2005) Deletion of etine therapy in fragile X syndrome, Developmental Brain FMR1 in Purkinje cells enhances parallel fiber LTD, enlarges spines, Dysfunction, 7, 164. and attenuates cerebellar eyelid conditioning in fragile X syndrome, Neuron, 47, 339–352. Hagerman, R. J., Miller, L. J., McGrath-Clarke, J. et al. (2002) Influence of stimulants on electrodermal studies in Fragile X syndrome, Kooy, R. F., Reyniers, E., Verhoye, M. et  al. (1999) Neuroanatomy of the Microscopy Research and Technique, 57, 168–173. fragile X knockout mouse brain studied using in vivo high resolution magnetic resonance imaging, European Journal of Human Genetics, Hagerman, R. J., Berry-Kravis, E., Kaufmann, W. E. et al. (2009) Advances 7, 526–532. in the Treatment of Fragile X Syndrome, Pediatrics, 123, 378–390. Krueger, D. D., Osterweil, E. K., Chen, S. P. et al. (2011) Cognitive dysfunc- Hagerman, R., Hoem, G. and Hagerman, P. (2010) Fragile X and autism: tion and prefrontal synaptic abnormalities in a mouse model of Intertwined at the molecular level leading to targeted treatments, fragile X syndrome, Proceedings of the National Academy of Molecular Autism, 1, 12. Sciences of the USA, 108, 2587–2592. Harrison, C. J., Jack, E. M., Allen, T. D. et al. (1983) The fragile X: a scanning Laggerbauer, B., Ostareck, D., Keidel, E. M. et  al. (2001) Evidence that electron microscope study, Journal of Medical Genetics, 20, fragile X mental retardation protein is a negative regulator of trans- 280–285. lation, Human Molecular Genetics, 10, 329–338. Henry, S. A., Lehmann-Masten, V., Gasparini, F. et al. (2002) The mGluR5 Lea, P. M., Movsesyan, V. A. and Faden, A. I. (2005) Neuroprotective activ- antagonist MPEP, but not the mGluR2/3 agonist LY314582, aug- ity of the mGluR5 antagonists MPEP and MTEP against acute excito- ments PCP effects on prepulse inhibition and locomotor activity, toxicity differs and does not reflect actions at mGluR5 receptors, Neuropharmacology, 43, 1199–1209. British Journal of Pharmacology, 145, 527–534. Huber, K. M., Kayser, M. S. and Bear, M. F. (2000) Role for rapid dendritic Levenga, J., Hayashi, S., de Vrij, F. M. S. et  al. (2011) AFQ056, a new protein synthesis in hippocampal mGluR-dependent long-term mGluR5 antagonist for treatment of fragile X syndrome, depression, Science, 288, 1254–1256. Neurobiology of Disease, 42, 311–317. Huber, K. M., Roder, J. C. and Bear, M. F. (2001) Chemical Induction of Li, J., Pelletier, M. R., Perez Velazquez, J. L. et al. (2002) Reduced cortical mGluR5- and protein synthesis-dependent long-term depres- synaptic plasticity and GluR1 expression associated with fragile X sion in hippocampal area CA1, Journal of Neurophysiology, 86, mental retardation protein deficiency, Molecular and Cellular 321–325. Neuroscience, 19, 138–151. Huber, K. M., Gallagher, S. M., Warren, S. T. et al. (2002) Altered synaptic plas- Loesch, D. Z., Huggins, R. M., Bui, Q. M. et al. (2003) Relationship of defi- ticity in a mouse model of fragile X mental retardation, Proceedings of cits of FMR1 gene specific protein with physical phenotype of fragile the National Academy of Sciences of the USA, 99, 7746–7750. X males and females in pedigrees: a new perspective, American Journal of Medical Genetics, 118A, 127–134. Incorpora, G., Sorge, G., Sorge, A. et  al. (2002) Epilepsy in fragile X syndrome, Brain and Development, 24, 766–769. Lu, Y. M., Jia, Z., Janus, C. et al. (1997) Mice lacking metabotropic gluta- mate receptor 5 show impaired learning and reduced CA1 long- Irwin, S. A., Galvez, R. and Greenough, W. T. (2000) Dendritic spine struc- term potentiation (LTP) but normal CA3 LTP, The Journal of tural anomalies in fragile-X mental retardation syndrome, Cerebral Neuroscience, 17, 5196–5205. Cortex, 10, 1038–1044. Manahan-Vaughan, D., Kulla, A. and Frey, J. U. (2000) Requirement of Jacquemont, S., Curie, A., Des Portes, V. et al. (2011) Epigenetic modifica- translation but not transcription for the maintenance of long-term tion of the FMR1 gene in fragile X syndrome is associated with depression in the CA1 region of freely moving rats, The Journal of differential response to the mGluR5 antagonist AFQ056, Sci Transl Neuroscience, 20, 8572–8576. Med, 64, 64ra1. Johnson, B. G., Wright, R. A., Arnold, M. B. et al. (1999) [3H]-LY341495 as a Martin, J. P. and Bell, J. (1943) A pedigree of mental defect showing novel antagonist radioligand for group II metabotropic glutamate sex-linkage, Journal of Neurology, Neurosurgery and Psychiatry, 6, (mGlu) receptors: characterization of binding to membranes of mGlu 154–157. 10 Bioscience Horizons • Volume 6 2013 Review article McBride, S. M. J., Choi, C. H., Wang, Y. et al. (2005) Pharmacological res- Sherman, S. L., Jacobs, P. A., Morton, N. E. et al. (1985) Further segrega- cue of synaptic plasticity, courtship behavior, and mushroom body tion analysis of the fragile X syndrome with special reference to defects in a Drosophila model of fragile X syndrome, Neuron, 45, transmitting males, Human Genetics, 69, 289–299. 753–764. Snyder, E. M., Philpot, B. D., Huber, K. M. et  al. (2001) Internalization of McLennan, Y., Polussa, J., Tassone, F. et  al. (2011) Fragile X syndrome, ionotropic glutamate receptors in response to mGluR activation, Current Genomics, 12, 216–224. Nature Neuroscience, 4, 1079–1085. Merenstein, S. A., Sobesky, W. E., Taylor, A. K. et  al. (1996) Molecular- Vanderklish, P. W. and Edelman, G. M. (2002) Dendritic spines elongate clinical correlations in males with an expanded FMR1 mutation, after stimulation of group 1 metabotropic glutamate receptors in American Journal of Medical Genetics, 64, 388–394. cultured hippocampal neurons, Proceedings of the National Academy of Sciences of the USA, 99, 1639–1644. Musumeci, S. A., Bosco, P., Calabrese, G. et al. (2000) Audiogenic seizures susceptibility in transgenic mice with fragile X syndrome, Epilepsia, Verkerk, A. J., Pieretti, M., Sutcliffe, J. S. et  al. (1991) Identification of a 41, 19–23. gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome, Cell, Oliet, S. H., Malenka, R. C. and Nicoll, R. A. (1997) Two distinct forms of 65, 905–914. long-term depression coexist in CA1 hippocampal pyramidal cells, Neuron, 18, 969–982. Weiler, I. J., Irwin, S. A., Klintsova, A. Y. et al. (1997) Fragile X mental retar- dation protein is translated near synapses in response to neu- Pieretti, M., Zhang, F. P., Fu, Y. H. et al. (1991) Absence of expression of the rotransmitter activation, Proceedings of the National Academy of FMR-1 gene in fragile X syndrome, Cell, 66, 817–822. Sciences of the USA, 94, 5395–5400. Pouladi, M. (2011) Fragile X syndrome therapy: to respond or not to respond Wilson, B. M. and Cox, C. L. (2007) Absence of metabotropic glutamate may be a matter of methylation, Clinical Genetics, 79, 508–510. receptor-Mediated plasticity in the neocortex of fragile X mice, Proceedings of the National Academy of Sciences of the USA, 104, Reddy, K. S. (2005) Cytogenetic abnormalities and fragile-x syndrome in 2454–2459. Autism Spectrum Disorder, BMC Med Genet, 6, 3. Yan, Q. J., Rammal, M., Tranfaglia, M. et  al. (2005) Suppression of two Sandler, A. (2005) Placebo effects in developmental disabilities: major fragile X syndrome mouse model phenotypes by the mGluR5 implications for research and practice, Mental Retardation antagonist MPEP, Neuropharmacology, 49, 1053–1066. and Developmental Disabililities Research Reviews, 11, 164–170. Zou, D., Huang, J., Wu, X. et al. (2007) Metabotropic glutamate subtype 5 Sherman, S. L., Morton, N. E., Jacobs, P. A. et  al. (1984) The marker (X) receptors modulate fear-conditioning induced enhancement of syndrome: a cytogenetic and genetic analysis, Annals of Human prepulse inhibition in rats, Neuropharmacology, 52, 476–486. Geneticsf, 48, 21–37. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bioscience Horizons Oxford University Press

mGlu5 as a potential therapeutic target for the treatment of fragile X syndrome

Bioscience Horizons , Volume 6 – Feb 11, 2013

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BioscienceHorizons Volume 6 • 2013 10.1093/biohorizons/hzt001 Review mGlu5 as a potential therapeutic target for the treatment of fragile X syndrome Lear Robertson Faculty of Biological Sciences, Institute of Membrane and Systems Biology, University of Leeds, Leeds LS2 9JT, UK. *Corresponding author: 1 Rochester Place, Charlbury, Chipping Norton, Oxfordshire OX7 3SF, UK. Email: lear.robertson@hotmail.co.uk Supervisor: Dr Steven Clapcote, Faculty of Biological Sciences, Institute of Membrane and Systems Biology, University of Leeds, Leeds LS2 9JT, UK. Fragile X syndrome (FXS) is the most common form of inherited mental retardation and the most common known cause of autism. It is caused by the expansion of a CGG trinucleotide repeat in the 5′ untranslated region of the fragile X mental retarda- tion 1 (FMR1) gene, which encodes the fragile X mental retardation protein (FMRP). FMRP negatively regulates group 1 (Grp 1) metabotropic glutamate receptor (mGlu a.k.a. mGluR) activity, and many FXS phenotypes are thought to be due to the over activity of the Grp 1 mGlu, mGlu5. This review evaluates the evidence for mGlu5 as a potential therapeutic target in the treat- ment of FXS. A 50% reduction in mGlu5 expression in Fmr1 knockout (KO) mice has been shown to reverse many FXS-relevant phenotypes including alterations in synaptic plasticity, increased dendritic spine density, increased basal hippocampal pro- tein synthesis, inhibitory avoidance extinction and susceptibility to audiogenic seizures. A negative modulator of mGlu5 may, therefore, be expected to have the same effect. In Fmr1 KO mice, Grp 1 mGlu antagonists, such as 2-methyl- 6-(phenylethynyl)pyridine (MPEP), fenobam and AFQ056, have been shown to reduce audiogenic seizures, reverse altered dendritic spine morphology, reduce excessive protein synthesis and improve behavioural abnormalities. MPEP, however, has failed to reverse altered long-term potentiation in the sensory neocortex or reduce machroorchidism. Clinical trials of mGlu5- negative modulators have had some positive outcomes but have had too few participants and were not performed over a long enough period to detect significant effects. Nevertheless, the prospects for development of mGlu5-negative modulators as FXS therapeutics are good and most research supports mGlu5 as a potential therapeutic target. Key words: fragile X syndrome, mGlu5, mGlu theory, Fmr1, FMRP Submitted on 6 September 2012; revised on 14 December 2012; accepted on 2 January 2013 Introduction et al., 1996). FMRP is a protein synthesis regulator at synapses, and functions as a translational repressor of target FXS is an inherited form of mental retardation first described mRNAs (Laggerbauer et al., 2001). It has also been hypoth- by Martin and Bell (1943) and was determined to be X-linked esized that FMRP plays a role in mRNA transportation along dominant with reduced penetrance after a large-scale segre- dendrites (Bassell and Warren, 2008). In FXS patients, there gation analysis by Sherman et al. (1984, 1985). Using scan- is a large decrease or complete silencing of the expression of ning electron microscopy, Harrison et al. (1983) found the the FMR1 gene, indicating that its loss of function is respon- X-chromosomal variant to be located at locus position sible for the syndrome. Xq27.3. Verkerk et al. (1991) became the first to clone the responsible gene: FMR1, which encodes the fragile X mental The causative mutation in FXS is a CGG expansion in the retardation protein (FMRP). FMRP is an RNA-binding pro- 5′ untranslated region of the FMR1 gene (Ashley et al., tein mainly present in the brain and testes and has been asso- 1993a). Normal individuals have a CGG trinucleotide repeat ciated with polyribosomes (Ashley et al., 1993b; Khandjian of between 6 and 54 (averaging 30 CGG units), while © The Author 2013. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Review Bioscience Horizons • Volume 6 2013 individuals with 55–200 have a fragile X premutation (Fu The mouse model et al., 1991). Individuals with >200 CGG repeats have the fragile X phenotype, females (XX) to a lesser extent due to The mouse Fmr1 gene is 97% homologous to the human compensation from the other, unaffected X chromosome. FMR1 gene (Ashley et al., 1993b). The first animal model of These CGG repeats lead to hypermethylation of the sur- FXS was created in 1994 by the Dutch Belgian fragile X rounding sequence including an upstream CpG island, caus- Consortium through the knock out (KO) of the Fmr1 gene in ing the silencing of FMR1 expression (Pieretti et al., 1991). mice (Bakker et al., 1994). FMRP was shown to be absent in FXS shows anticipation, which refers to the number of the the Fmr1 KO mice by western blotting and the phenotype trinucleotide repeats increasing from one generation to the appears consistent with, yet less severe than, human FXS; the next, meaning the risk of FXS increases in successive genera- mice had mild behavioural abnormalities, mild mental retar- tions (Fu et al., 1991). dation and machroorchidism. Most mouse models have been created in the same way: by KO of the Fmr1 gene and there- FXS is the most common inherited form of mental retar- fore do not include the expanded CGG trinucleotide repeat dation and is also responsible for 2–6% of autism cases, and so do not precisely model the causative mutation in FXS. making it the most common single gene mutation to cause The methylation state of the FMR1 promoter region depends autism (Reddy, 2005; Hagerman, Hoem and Hagerman, on the number of CGG repeats; Fmr1 KO mice only model 2010). Recent estimates of prevalence using DNA-based the fully methylated form of FXS, which represents only a diagnostic techniques revealed that FXS is present in ~1 in fraction of humans with FXS (Jacquemont et al., 2011). 4500 males and ~1 in 9000 females, with the premutation However, since KO of Fmr1 and the silencing of FMR1 by present in ~1 in 1000 males and ~1 in 400 females, it is pres- CGG expansion in human FXS both result in the lack of ent in every ethnic group (Crawford, Acuña and Sherman, expression of FMRP the mouse model is sufficiently similar 2001). to be useful in FXS research. In males, FXS is characterized by moderate-to-severe men- Mouse models of FXS do not replicate the anatomical tal retardation and autistic behaviours (Merenstein et al., abnormalities found in the brains of FXS patients. The size of 1996). There is also a developmental delay, and the majority the posterior vermis of the cerebellum in humans suffering of males suffer from attention deficit hyperactivity disorder from FXS is significantly reduced, but there is no difference in (ADHD) (Hagerman et al., 2009). Another neurological phe- the size of the posterior vermis in Fmr1 KO mice when com- notype present in 10–20% of boys with FXS and ~5% of pared with WT littermates (Kooy et al., 1999). However, girls is epilepsy, usually first occurring between the ages of 4 Fmr1 KO mice and humans with FXS both have more dense and 10 and ending later in childhood (Berry-Kravis, 2002; dendritic spines which tend to be ‘immature’ (long and thin) Incorpora et al., 2002). A connective tissue dysplasia results in appearance, but there is disagreement in the literature as to in the physical symptoms of FXS (narrow face, large ears, the precise phenotype (Irwin, Galvez and Greenough, 2000; machroorchidism and hyperextensible fingers) and it has Snyder et al., 2001; Vanderklish and Edelman, 2002). been hypothesized that hypothalamic dysfunction leads to the development of short bodies and limbs (Loesch et al., One reason that this mouse model of FXS has been 2003). In childhood, ~10% males with FXS develop Prader- accepted so readily is the broad range of behavioural pheno- Willi phenotype that consists of obesity and hyperphagia (de types that it reproduces (Bakker et al., 1994). FXS is a com- Vries et al., 1993; McLennan et al., 2011). plex disease with various neurological symptoms, many of which are expressed in the KO mice, including seizure sus- There is no cure for FXS, and the core symptom of mental ceptibility, social behaviour, sensitivity to stimuli and cogni- retardation is currently untreatable. However, some other tive performance (Musumeci et al., 2000; Dölen et al., 2007; symptoms of FXS may be improved pharmacologically or by de Vrij et al., 2008). Behavioural phenotype assays have thus behavioural interventions. α-adrenergic receptor agonists are far shown small effects in Fmr1 KO mice compared with used to treat the ADHD and selective serotonin reuptake humans with FXS. This may be because conventionally hip- inhibitors have been shown to be successful for treating anx- pocampus-dependent learning tasks, such as the Morris iety in over 50% of cases (Hagerman, Murphy and water maze, have been used, whereas a recent study indicates Wittenberger, 1988; Hagerman et al., 1994, 2002). A single that prefrontal cortex-dependent learning tasks could reveal anticonvulsant is often used to treat the epilepsy and antipsy- larger effects of Fmr1 KO (Krueger et al., 2011). chotics can help stabilize mood (Berry-Kravis and Potanos, 2004; Hagerman et al., 2009). There are no pharmaceuticals to treat the Prada-Willi phenotype, so the patient’s diet and The mGlu theory of FXS exercise must be regulated (Balko, 2005; Chen et al., 2007). Due to the lack of cure there is a clear need for new and bet- Mouse models have been instrumental in many break- ter therapies for FXS. This review evaluates the evidence for throughs in FXS research, revealing FXS as a disorder of metabotropic glutamate receptor 5 (mGlu5) as a potential long-term depression (LTD) and long-term potentiation therapeutic target in the treatment of FXS, and discusses how (LTP) in the cerebellum, the hippocampus and the prefrontal this might be achieved. cortex (Huber et al., 2002; Li et al., 2002; Koekkoek et al., 2 Bioscience Horizons • Volume 6 2013 Review article −/Y +/− 2005). The activation of both the N-methyl-d-aspartate reduction in mGlu5 [Fmr1 ; Grm5 ] (Lu et al., 1997). (NMDA) receptors and the Group (Grp) 1 mGlus results in Reducing mGlu5 expression by 50% in Fmr1 KO mice led to decreased postsynaptic 2-amino-3-(5-methyl-3-oxo-1,2-oxa- the rescue of various FXS-related phenotypes, including syn- zol-4-yl)propanoic acid (AMPA) and NMDA receptors due aptic plasticity alterations, increased dendritic spine density, to internalization (Oliet, Malenka and Nicoll, 1997). One increased basal hippocampal protein synthesis, exaggerated major mechanical dissimilarity between these two pathways inhibitor avoidance extinction and audiogenic seizure suscep- is that the mGlu-dependent LTD involves immediate mRNA tibility. This evidence supports the mGlu theory as well as the translation at the synapse, whereas NMDA-dependent LTD notion that pharmaceutical inhibition of mGlu5 could have a only requires mRNA translation if the LTD continues for similar effect. However, the 50% reduction in mGlu5 expres- more than a few hours (Huber, Kayser and Bear, 2000; sion in Fmr1 KO mice did not reverse the machroorchidism Manahan-Vaughan, Kulla and Frey, 2000). mGlu-dependent found in FXS (Dölen et al., 2007), indicating that mGlu5 does LTD is also irreversible and entails the loss of the synapse, not control testicle size. This raises the possibility that there unlike NMDA-dependent LTD which can be readily reversed may be other FXS phenotypes that are not rescued by reduced (Oliet, Malenka and Nicoll, 1997; Snyder et al., 2001). mGlu5 activity. Postnatal pharmacological inhibition of mGlu5 may not have the same effect as genetic inhibition of The study of FMRP in mGlu-dependent LTD was started mGlu5 in FXS patients and mouse models because sufficient when it was discovered that the activation of mGlus pro- damage may have been done already during prenatal develop- moted the synthesis of FMRP in synaptoneurosomes (Weiler ment to make the FXS phenotype irreversible. et al., 1997). mGlu-dependent LTD in Fmr1 KO mice was significantly increased compared with wild-type (WT) litter - mates but there was no difference in NMDA-dependent LTD, Pharmacological inhibition of mGlu5 indicating that the phenotype is exclusively linked to mGlu- dependent synaptic plasticity (Huber et al., 2002). mGlu5 is in mouse models of FXS the most prominent Grp 1 mGlu in the forebrain and thus it One behavioural phenotype that is present in both humans was hypothesized that mGlu5 activation stimulates the syn- with FXS and Fmr1 KO mice is heightened sensitivity to sen- thesis of proteins which enhance LTD, and FMRP inhibits sory stimuli. This has been shown to be related to mGlu sig- further synthesis of these proteins, therefore controlling the nalling and can be tested using prepulse inhibition (PPI), a LTD (Fig. 1A) (Bear, Huber and Warren, 2004). Excessive technique in which a reduced startle response is recorded synthesis of these proteins, as occurs in FXS, leads to inter- from a stimulus after a weaker prestimulus (Grauer and nalization of AMPA and NMDA receptors, causing the for- Marquis, 1999). de Vrij et al. (2008) studied PPI of acoustic mation of abnormally long and thin dendritic spines (Fig. 1B) startle responses in Fmr1 KO and WT mice. In WT mice, PPI (Irwin et al., 2000; Snyder et al., 2001; Vanderklish and was 73%, whereas it was 30% in Fmr1 KO mice, evidence of Edelman, 2002). the heightened sensitivity in the KO mice. Fmr1 KO mice treated with 20 mg/kg of the mGlu5 inhibitor 2-methyl-6- Mouse models and the mGlu theory (phenylethynyl)pyridine (MPEP) 30 mins before the experi- ment displayed PPI of 70%, an apparent rescue of the mGlu-dependent LTD was implicated in FXS by Huber et al. phenotype (de Vrij et al., 2008). However, Frankland et al. (2002). Paired-pulse facilitation and 3,5-dihydroxyphenylg- (2004) recorded increased PPI in Fmr1 KO mice compared lycine (DHPG) were independently used to induce LTD in with WT mice. This variation could be due to different exper- separate hippocampal slices of Fmr1 KO mice and WT lit- imental techniques, since de Vrij et al. (2008) measured the termates. The NMDA antagonist D-APV was used to prevent eyelid startle response, whereas Frankland et al. (2004) mea- NMDA-dependent LTD and so the experiment selectively sured the whole-body startle response. Moreover, while de recognized mGlu-dependent LTD. In the case of both the Vrij et al. (2008) found that MPEP increased PPI in WT mice, paired-pulse facilitation and DHPG, mGlu-dependent LTD this effect on PPI has not been observed in rats treated with was significantly enhanced in Fmr1 KO mice but not in WT MPEP (Henry et al., 2002; Zou et al., 2007). littermates, indicating that mGlu5 is overactive in FXS. MPEP has also been shown to reduce audiogenic seizures, This finding led to the hypothesis that the inhibition of which are characteristic of Fmr1 KO mice (Yan et al., 2005). mGlu5 may be a potential therapy for FXS (Bear, Huber and MPEP reduced audiogenic seizures in both Fmr1 KO and WT Warren, 2004). This was further investigated by Dölen et al. mice with three different genetic backgrounds (FVB/NJ, (2007), who generated Fmr1 KO mice with a 50% reduction C57BL/6J and an F1 hybrid of the two), although the dose in mGlu5 expression. This is possible because the genes for was larger in the KO mice. Application of MPEP also rescued FMRP and mGlu5 (FMR1 and GRM5) have single functional the open field phenotype, MPEP-treated Fmr1 KO mice spent homologues in mice (Fmr1 and Gmr5). By crossing Fmr1 KO as much time as WT mice in the centre of the open field mice with Gmr5 KO mice, offspring were produced with arena. These results support mGlu5 inhibition as a potential Fmr1 KO and 50% reduced mGlu5 expression. Complete KO therapy in FXS. Importantly, Yan et al. (2005) also discov- of mGlu5 causes impaired brain function and so it was impor- ered that mice can develop a tolerance to MPEP with repeat tant to study Gmr5 heterozygous mice with only a 50% 3 Review Bioscience Horizons • Volume 6 2013 Figure 1. Models representing the mGlu theory of FXS. (A) A model of the effect of mGlu5 activation and FMRP on mRNA translation at neuron synapses. mGlu5 activation causes AMPA receptor recycling and mRNA translation. This mRNA translation causes AMPA receptor internalization and is negatively regulated by FMRP. In FXS, FMRP is not present and so the mRNA translation is in excess as is the AMPA receptor internalization. (B) A model of the effect of FXS on dendritic spine shape. As AMPA and NMDA receptors are lost, the dendritic spines take on an ‘immature’ appearance (long and thin). Taken from Bear, Huber and Warren (2004). administration of the drug, which could prove to be a prob- case, MPEP may impair auditory acuity but would not be lem when developing mGlu5 antagonists as FXS treatments. targeting the core symptoms of FXS. Although the Yan et al. (2005) study appeared to show The de Vrij et al. (2008) study also determined the effect of that MPEP reverses two FXS-relevant phenotypes in Fmr1 mGlu5 antagonists MPEP and fenobam on dendritic spine KO mice, there could be another explanation for its effects. It morphology in Fmr1 KO mice. The morphology and density is conceivable that MPEP is reducing excitatory glutaminer- of dendritic spines has become a common test for the efficacy gic activity in the auditory pathway, thereby reducing the of pharmaceuticals in FXS. Although it is recognized that effect of the stimulus causing the seizures. If this were the there is some alteration in dendritic spine morphology in FXS 4 Bioscience Horizons • Volume 6 2013 Review article and Fmr1 KO mice, the literature is not in agreement on the mice, but in the KO mice the density of filopodia was signifi- specific alterations. It is generally accepted that the ratio of cantly greater. After treatment with the mGlu5 agonists ‘immature’ long thin spines (filopodia) to ‘mature’ mush- MPEP or fenobam, the ratio of normal spines to filopodia room shaped spines is greater in FXS and in Fmr1 KO mice matched those of the WT mice. The results of this study con- and that the density of the spines increased (Fig. 2) (Irwin, form to the predictions made by Dölen et al. (2007), who Galvez and Greenough, 2000). However, studies have pro- rescued the increased dendritic spine density by reducing duced results varying from reduced spine density in hippo- mGlu5 expression. campal cultures of Fmr1 KO mice (Braun and Segal, 2000) to The mGlu theory postulates that this altered spine mor- hugely dense filopodia (3–5 filopodia per 10 µm) (Antar phology is due to increased protein synthesis, which is regu- et al., 2006). These differing results could arise from varying lated by FMRP and stimulated by mGlu5 activation. FMRP definitions of ‘immature spines’ and the subjectivity behind binds with up to 4% of the brain’s mRNA in dendrites, their identification. To make the classification of these filopo- including its own, and suppresses its translation to protein dia more objective, de Vrij et al. (2008) considered all spines (Weiler et al., 1997; Laggerbauer et al., 2001). Translation of with length greater than the width to be filopodia and any FMRP therefore acts as a negative feedback mechanism to width greater than or equal to the length to be mature spines. stop excess translation caused by mGlu5 activation (Huber In this way, de Vrij et al. (2008) found that the density of et al., 2002) (Fig. 1A). Due to the lack of expression of FMRP mature spines was the same in both WT mice and Fmr1 KO in FXS, there is no negative feedback. The function of FMRP in mRNA translational regulation is incredibly complex and diverse, with much controversy surrounding its precise role. FMRP has been shown to be a translational activator as well as a repressor; it plays a part in translation initiation and elongation as well as being associated with the RNA-induced silencing complex. Many of the mRNAs that FMRP binds to, such as MAP1B, are factors in spine morphology (Gross, Berry-Kravis and Bassell, 2012). Aschrafi et al. (2005) observed decreased mRNA granules (shown to contain FMRP by western blot of rat brain lysate) in the brains of Fmr1 KO mice compared with WT. When treated with 35 mg/kg of MPEP, the size of the mRNA gran- ules were significantly increased in both Fmr1 KO and WT mice. This suggests that granules are stabilized by FMRP and destabilized by mGlu5 activity. This study does not directly measure protein synthesis; the link between mRNA granules and level of protein synthesis is only hypothetical. It is unclear why Aschrafi et al. (2005) performed a western blot of brain lysate from rats to prove the presence of FMRP in mRNA granules: the study does not show that FMRP is present in mRNA granules in mice brains. This increased protein synthesis leads to altered synaptic plasticity. In Fmr1 KO mice, the most characterized form of altered synaptic plasticity is enhanced mGlu-dependent LTD in the Shaffer collaterals of the CA1 region of the hippocam- pus (Huber, Roder and Bear, 2001; Huber et al., 2002). To date, no studies have investigated the effects of mGlu5-nega- tive modulation on LTD anywhere in the brain. However, the effect of the Grp 2 mGlu antagonist LY341495 on mGlu- dependent LTD in Fmr1 KO mice has been researched in CA1 neurons (Choi et al., 2011), the rationale being that antago- nism of DmGluA, the Drosophila flies sole mGlu homologue, has been shown to rescue memory and social interaction (McBride et al., 2005). DmGluA has both Grp 1 and 2 mGlu activity. Chronic treatment with the Grp 2 mGlu antagonist Figure 2. Spine morphologies of Golgi-impregnated neurons. (A) LY341495 for 8 weeks rescued DHPG-induced LTD in adult Dendritic spines of human affected with FXS. (B) Dendritic spines of a Fmr1 KO mice, but increased DHPG-induced LTD in WT human without FXS. The density of long thin spines is much greater in mice. The reason for the contrasting effects in Fmr1 KO and (A) than in (B). Taken from Irwin, Galvez and Greenough, (2000). 5 Review Bioscience Horizons • Volume 6 2013 WT mice is unknown but Choi et al. (2011) hypothesized that function that mGlu5-negative modulators would rescue may it could be due to some sort of compensation in the system. be the regulation of mGlu5 activity. However, LY341495 is not completely selective for Grp 2 MPEP is not a specific inhibitor of mGlu5, and can inhibit mGlus, as it is also a Grp 3 and a Grp 1 mGlu antagonist. NMDA receptors when at high concentrations (Lea, Movsesyan LY341495 has a 1000-fold greater affinity for Grp 2 mGlus and Faden, 2005), thus raising the possibility that MPEP than Grp 1, and 10-fold greater for Grp 2 than for Grp 3 treatment could potentially have adverse effects. Most stud- (Kingston et al., 1998). The dose was also kept at the lowest ies have tried to use MPEP at concentrations low enough not level previously shown to reverse the in vivo effects of a Grp 2 to affect NMDA activity, but it is conceivable that some con- mGlu agonist (Johnson et al., 1999). Nevertheless, it cannot clusions drawn from research involving MPEP could be due be ruled out that the antagonistic effect on the Grp 1 or Grp 3 to its NMDA-inhibiting action rather than its ability to mGlus did not cause the phenotype rescue. inhibit mGlu5. MPEP cannot be used as a therapy in humans In contrast to LDP, LTP is reduced in the sensory neocor- with FXS owing to its toxicity. It also has a very short half-life tex of Fmr1 KO mice but not in the hippocampus (Fig. 3) (~1 h in C57BL/6J mice, Anderson et al., 2003) meaning that (Li et al., 2002). Wilson and Cox (2007) showed that in the administration of the drug would have be more frequent vitro LTP in the visual neocortex in WT mice is not only than is practicable. NMDA-dependent but primarily due to mGlu activity. They AFQ056 is a drug developed by Novartis which could proposed that the reduced LTP measured in the visual cortex potentially be used to treat FXS in humans since it does not of Fmr1 KO mice is due to reduced mGlu5 activity. When have the same drawbacks as MPEP, such as toxicity and the mGlu5 inhibitor MPEP was applied to the visual cortex short half-life. Levenga et al. (2011) tested the effect of of Fmr1 KO mice, there was no significant difference in LTP AFQ056 on the spine morphology and PPI of Fmr1 KO between MPEP-treated and control Fmr1 KO mice. This mice. AFQ056 was shown to restore PPI in Fmr1 KO mice suggests that mGlu5-mediated synaptic plasticity is in deficit from 22 to 48% and also increased PPI in WT mice. AFQ056 in the visual cortex, in contrast to the hippocampus where also affected spine morphology: when administered to Fmr1 mGlu5-mediated synaptic plasticity is in excess. If this is the KO mice in three different concentrations (10 nm, 1 µ M and case, it poses serious problems for mGlu5 as a potential ther- 10 µ M), the hippocampal dendritic spine length decreased. apeutic target; since it shows that the relationship between Interestingly, AFQ056 increased spine density and decreased FMRP and mGlu5 is more complicated than originally spine width. This is in contrast to the results of the in vivo thought. It also implies that FMRP-regulated events may be study by Dölen et al. (2007), in which a decrease in the spine specific to particular brain areas, rendering mGlu5 inhibi- density and an increase in the spine width was observed tion useless or perhaps even harmful in some areas of the when mGlu5 expression was reduced by 50% in Fmr1 KO brain. mice. As a relatively new drug, very little research has been Yan et al. (2005) postulated that mGlu5-negative modula- conducted using AFQ056. There have not yet been any stud- tors, such as MPEP, will only have an effect on phenotypes ies published that confirm AFQ056 as a selective mGlu5 caused by FMRP-independent signalling and may have no antagonist as it is described by Levenga et al. (2011). Much effect on FMRP-dependent signalling, thus, some FXS phe- more research is needed before it can be considered a poten- notypes will remain (Fig. 4). Essentially, the only FMRP tial therapy. Figure 3. Graph showing the LTP response to tetanic stimulation in WT and in Fmr1 KO mice. Taken from deep layers of the visual neocortex in the presence of low concentration of the GABA antagonist bicuculline. LTP is significantly reduced in the Fmr1 KO mice compared with the WT mice over 60 min of tetanic stimulation. MPEP did not rescue the reduced LTP in Fmr1 KO mice (data not shown). Taken from Wilson and Cox (2007); Copyright 2007 National Academy of Sciences, USA. 6 Bioscience Horizons • Volume 6 2013 Review article conducted on 30 individuals with FXS (Jacquemont et al., 2011). Individuals were screened to determine the extent of the methylation of their FMR1 promoter. Interestingly, only individuals with a fully methylated promoter and no FMR1 mRNA detected in the blood showed significant improve- ments in comparison with the control group when measured using various behavioural rating scales. This suggests that screening for full methylation could determine which patients would benefit from mGlu5 antagonists. The response to treatment in patients with partial methyla- tion of the FMR1 promoter was varied. Jacquemont et al. (2011) propose that this variation in response is due to differ- ence in methylation states and therefore to different degrees of mGlu5 hyperactivity (Fig. 5). The baseline scores of the behav- ioural rating scales indicate a more severe phenotype in the fully methylated participants which supports this theory; however, this difference is not statistically significant. The lack of correla- tion between FMR1 mRNA in the blood and response to AFQ056 casts doubt over this hypothesis, although it could be explained by variance in tissue-specific methylation or variance in the translation of FMR1 mRNA to FMRP. An assessment of Figure 4. A diagram of MPEP effect on mGlu5 signalling. mGlu5 signalling: FMRP independent (A), and FMRP dependent (B). Negative FMRP levels may have helped draw conclusions as to why modulators of mGlu5 may only have effect on A and not on B. This is there was so much variation in response amongst the partially because by negatively modulating mGlu5, FMRP’s inhibitory effect on methylated subpopulation but no such analysis was carried mGlu5 signalling (A) will be mimicked, whereas the FMRP itself will still out. The small number of trial patients (seven with fully meth- not be present and therefore no other functions of FMRP (B) will be ylated FMR1 promoters) makes it difficult to draw any solid rescued. conclusions from this study. Future trials with more partici- pants may offer clues to the mechanism behind why fully meth- Clinical trials with mGlu5-negative ylated participants responded more consistently to treatment. modulators in FXS Jacquemont et al. (2011) chose to use behavioural response to measure the efficacy of AFQ056. Measurement of change In recent years, the positive results of studies into mGlu5- negative modulators in preclinical animal models has led to the development of pharmaceuticals and clinical trials in humans with FXS that target mGlu5, including fenobam, AFQ056 (Novartis), STX107 (Seaside Therapeutics) and RG7090 (Hoffmann-LaRoche). A pilot open label, single-dose trial of fenobam was con- ducted on 12 subjects (6 male, 6 female) with FXS (Berry- Kravis et al., 2009). The main aim of the experiment was to determine the safety of the drug and identify any significant adverse effects, of which there was none. Each patient received a single dose of fenobam (between 50 and 150 mg) and was then screened for vital signs and side effects during the following 6 h. 50% of the patients had amended PPI after drug administration ranging from 23.7 to 44.2% improve- ment. It is known that the placebo effect is exaggerated in individuals with mental retardation (Sandler, 2005) and so Figure 5. Methylation state of the FMR1 promoter affects the efficacy the open label nature of this trial may have some influence on of AFQ056. Schematic showing the influence that the methylation the results. The results also suffered due to the limited num- state of the FMR1 promoter has over the efficacy of AFQ056 in FXS ber of patients in the trial and the fact that they were only patients. Participants with fully methylated promoters (only given a single dose. However, the lack of adverse effects is methylated DNA was detected) experienced a behavioural promising for future trials. improvement in response to AFQ056, whereas participants with partially methylated promoters (methylated and non-methylated DNA A double-blind, two-treatment, two-period crossover trial was detected) had varying responses and no statistically significant of the subtype-selective mGlu5 inhibitor AFQ056 was improvement in behaviour. Taken from Pouladi (2011). 7 Review Bioscience Horizons • Volume 6 2013 in PPI or eye tracking would have been a more objective way human disease. In the mouse models, the behavioural pheno- to quantify the response. Also, to see a developmental type is less obvious and the symptoms much milder than in improvement in the participants rather than just symptomatic human FXS. Overall, the prospect for the development of improvements, future trials will have to be conducted over mGlu5-negative modulators as a therapy appears good, as is significantly longer time periods, particularly in older patients. evident from the clinical trials now underway. Currently, there are other mGlu5-negative modulators One important question in need of answering is whether or such as STX107 and RG7090, which are undergoing clinical not the effect of FXS during prenatal development is reversible trials but the result have yet to be released. by pharmaceuticals. The application of mGlu5-negative mod- ulators is unlikely to be a practical option as the average age of diagnosis is at ~35–37 months in males and later in females Discussion (Bailey et al., 2009). In most models, it has been shown that at least some of the phenotypes are reversed by treatment with Much of the evidence in preclinical animal models of FXS mGlu5-negative modulators in adulthood. The clinical trials indicates that mGlu5-negative modulators may eventually be during prenatal development that have been performed so far used therapeutically. The studies using mouse models are tell us very little about the long-term effects of mGlu5-negative summarized in Table 1. There are minor inconsistencies modulators. Future trials need to be carried out over a much within these studies, many of which could be due to differing longer time period and with many more participants to find experimental techniques. Taken collectively, however, these out to what extent these drugs can be beneficial. works tend to support the mGlu theory put forward by Bear, Huber and Warren (2004) but do not paint a complete pic- Twenty-four of the 30 participants in the Jacquemont ture. Some results indicate that Grp 2 mGlus may also be et al. (2011) study experienced adverse effects, mostly fatigue implicated in the mGlu theory (Wilson and Cox, 2007; Choi and headaches but some suffered from hyperlipasemia, et al., 2011). The major limitation within the preclinical hyperamylasemia, increased hepatic enzymes and increased studies is the fundamental problem of modelling human blood creatinine phosphokinase. At higher doses these diseases in animals. The validity of the models depends on adverse effects may become more severe and as this was a the extent to which the animal disease is analogous to the relatively small trial there may be other more dangerous side Table 1. Summary of studies of mGlu5-negative modulation in Fmr1 KO mice Therapy Reversed phenotype Target Study Comments/discrepancies Genetic rescue; Altered synaptic plasticity, mGlu5 Dölen et al. 50% reduction in the mGlu5 expression was unable to 50% reduction dendritic spine density, basal (2007) rescue machroorchidism in mGlu5 hippocampal protein synthesis, expression inhibitor avoidance extinction, audiogenic seizures MPEP Decreased PPI of acoustic startle Grp 1 mGlus de Vrij et al. Inconsistent with the findings of Frankland et al. (2004) response (2008) who observed increased PPI in Fmr1 KO mice compared with WT. Hugely varying alterations in spine morphol- ogy was observed in Fmr1 KO mice (Braun and Segal, 2000; Antar et al., 2006) MPEP, fenobam Increased ratio of filopodia to Grp 1 mGlus de Vrij et al. As above. mature dendritic spines (2008) MPEP Audiogenic seizures, centre field Grp 1 mGlus Yan et al. Reduction in auditory seizures could be due to reduced behaviour (2005) glutaminergic activity in the auditory pathway decreasing the effect of the stimulus. MPEP was also unable to rescue increased size of testis MPEP Excessive protein synthesis Grp 1 mGlus Aschrafi No evidence that FMRP is present in mRNA granules in et al. (2004) mouse brains. Link between the mRNA granule size and the level of protein synthesis is only hypothetical LY341495 Increased DHPG-induced LTD in Grp 2 mGlus Choi et al. LY341495 has a very small antagonistic effect on Grps the CA1 region of the (2011) 1 and 3 mGlus hippocampus MPEP None Gp 1 mGlus in Wilson and MPEP did not rescue excess LTP in the sensory sensory neocortex Cox (2007) neocortex AFQ056 Decreased PPI, increased spine Grp 1 mGlus Levenga AFQ056 increased spine density and decreased spine length et al. (2011) width which is in contrast to Dölen et al. (2007) in which the decreased mGlu5 expression did the opposite 8 Bioscience Horizons • Volume 6 2013 Review article Bakker, C. E., Verheij, C., Willemsen, R. et al. (1994) Fmr1 knockout mice: effects that did not present themselves. The results of the a model to study fragile X mental retardation, The Dutch-Belgian Wilson and Cox (2007) study indicate a variance in the Fragile X Consortium, Cell, 78, 23–33. mGlu5 activity across different brain regions; this is a poten- tial problem with regard to the therapeutic use of mGlu5- Balko, K. (2005) Red Yellow Green System for Weight Management. negative modulators. Specifically mGlu5 inhibition has been Toronto: Ontario Prader-Willi Syndrome Association. shown to have no effect on the increased LTP in the visual cortex and may have a negative effect by decreasing visual Bassell, G. J. and Warren, S. T. (2008) Fragile X Syndrome: loss of local cortex LTD (Wilson and Cox, 2007). mRNA regulation alters synaptic development and function, Neuron, 60, 201–214. Inhibition of mGlu5 does not offer a complete cure for FXS, since some symptoms such as machroorchidism have Bear, M. F., Huber, K. M. and Warren, S. T. (2004) The mGluR theory proved to be resistant to mGlu5-negative modulation and of  fragile X mental retardation, Trends in Neuroscience, 27, there may be other refractory symptoms yet to be identified 370–377. (Dölen et al., 2007). Perhaps inhibition of mGlu5 will help to Berry-Kravis, E. (2002) Epilepsy in fragile X syndrome, Developmental control many of the symptoms and, along with other drugs, Medicine and Child Neurology, 44, 724–728. significantly improve the quality of life of the patient and the carers. With further research into mGlu5-negative modula- Berry-Kravis, E. and Potanos, K. (2004) Psychopharmacology in fragile X tors in animal models and through clinical trials, their full syndrome—present and future, Mental Retardation and therapeutic potential will be revealed. Developmental Disabililities Research Reviews, 10, 42–48. Berry-Kravis, E., Hessl, D., Coffey, S. et al. (2009) A pilot open label, single Acknowledgments dose trial of fenobam in adults with fragile X syndrome, Journal of Medical Genetics, 46, 266–271. Thanks to Steve Clapcote for his help and advice, to Peter Wright for editing and to Francesca Deane for motivation. Braun, K. and Segal, M. (2000) FMRP involvement in formation of syn- apses among cultured hippocampal neurons, Cerebral Cortex, 10, 1045–1052. Author biography Chen, C., Visootsak, J., Dills, S. et  al. (2007) Prader-Willi syndrome: an I graduated from the University of Leeds in July 2012 with a update and review for the primary pediatrician, Clinical Pediatrcs, degree in neuroscience. My particular interests are neurologi- 46, 580–591. cal disorders and the development of therapies for them. I am currently applying for postgraduate study. Choi, C. H., Schoenfeld, B. P., Bell, A. J. et  al. (2011) Pharmacological reversal of synaptic plasticity deficits in the mouse model of fragile X syndrome by group II mGluR antagonist or lithium treatment, References Brain Research, 1380, 106–119. Anderson, J. J., Bradbury, M. J., Giracello, D. R. et al. (2003) In vivo recep- Crawford, D. C., Acuña, J. M. and Sherman, S. L. (2001) FMR1 and the tor occupancy of mGlu5 receptor antagonists using the novel radio- fragile X syndrome: human genome epidemiology review, Genetics ligand [3H]3-methoxy-5-(pyridin-2-ylethynyl)pyridine), European in Medicine, 3, 359–371. Journal of Pharmacology, 473, 35–40. de Vries, B. B., Fryns, J. P., Butler, M. G. et al. (1993) Clinical and molecular Antar, L. N., Li, C., Zhang, H. et al. (2006) Local functions for FMRP in axon studies in fragile X patients with a Prader-Willi-like phenotype, growth cone motility and activity-dependent regulation of filopodia Journal of Medical Genetics, 30, 761–766. and spine synapses, Molecular and Cellular Neuroscience, 32, 37–48. de Vrij, F. M. S., Levenga, J., van der Linde, H. C. et  al. (2008) Rescue of Aschrafi, A., Cunningham, B. A., Edelman, G. M. et al. (2005) The fragile X behavioral phenotype and neuronal protrusion morphology in mental retardation protein and group I metabotropic glutamate Fmr1 KO mice, Neurobiology of Disease, 31, 127–132. receptors regulate levels of mRNA granules in brain, Proceedings of the National Academy of Sciences of the USA, 102, 2180–2185. Dölen, G., Osterweil, E., Shankaranarayana Rao, B. S. et  al. (2007) Correction of fragile X syndrome in mice, Neuron, 56, 955–962. Ashley, C. T., Wilkinson, K. D., Reines, D. et al. (1993a) FMR1 protein: con- served RNP family domains and selective RNA binding, Science, 262, Frankland, P. W., Wang, Y., Rosner, B. et  al. (2004) Sensorimotor gating 563–566. abnormalities in young males with fragile X syndrome and Fmr1- knockout mice, Molecular Psychiatry, 9, 417–425. Ashley, C. T., Sutcliffe, J. S., Kunst, C. B. et al. (1993b) Human and murine FMR-1: alternative splicing and translational initiation downstream Fu, Y. H., Kuhl, D. P., Pizzuti, A. et al. (1991) Variation of the CGG repeat at of the CGG-repeat, Nature Genetics, 4, 244–251. the fragile X site results in genetic instability: resolution of the Sherman paradox, Cell, 67, 1047–1058. Bailey, D. B., Raspa, M., Bishop, E. et  al. (2009) No change in the age of diagnosis for fragile X syndrome: findings from a National Parent Grauer, S. M. and Marquis, K. L. (1999) Intracerebral administration of Survey, Pediatrics, 124, 527–533. metabotropic glutamate receptor agonists disrupts prepulse 9 Review Bioscience Horizons • Volume 6 2013 inhibition of acoustic startle in Sprague-Dawley rats, Psycho- receptor subtype expressing cells, Neuropharmacology, 38, 1519– pharmacology (Berl.), 141, 405–412. 1529. Gross, C., Berry-Kravis, E. M. and Bassell, G. J. (2012) Therapeutic strate- Khandjian, E. W., Corbin, F., Woerly, S. et  al. (1996) The fragile X mental gies in fragile X syndrome: dysregulated mGluR signaling and retardation protein is associated with ribosomes, Nature Genetics, beyond, Neuropsychopharmacology, 37, 178–195. 12, 91–93. Hagerman, R. J., Murphy, M. A. and Wittenberger, M. D. (1988) A con- Kingston, A. E., Ornstein, P. L., Wright, R. A. et  al. (1998) LY341495 is a trolled trial of stimulant medication in children with the fragile X nanomolar potent and selective antagonist of group II metabo- syndrome, American Journal of Medical Genetics, 30, 377–392. tropic glutamate receptors, Neuropharmacology, 37, 1–12. Hagerman, R. J., Fulton, M. J., Leaman, A. et al. (1994) A survey of fluox - Koekkoek, S. K. E., Yamaguchi, K., Milojkovic, B. A. et al. (2005) Deletion of etine therapy in fragile X syndrome, Developmental Brain FMR1 in Purkinje cells enhances parallel fiber LTD, enlarges spines, Dysfunction, 7, 164. and attenuates cerebellar eyelid conditioning in fragile X syndrome, Neuron, 47, 339–352. Hagerman, R. J., Miller, L. J., McGrath-Clarke, J. et al. (2002) Influence of stimulants on electrodermal studies in Fragile X syndrome, Kooy, R. F., Reyniers, E., Verhoye, M. et  al. (1999) Neuroanatomy of the Microscopy Research and Technique, 57, 168–173. fragile X knockout mouse brain studied using in vivo high resolution magnetic resonance imaging, European Journal of Human Genetics, Hagerman, R. J., Berry-Kravis, E., Kaufmann, W. E. et al. (2009) Advances 7, 526–532. in the Treatment of Fragile X Syndrome, Pediatrics, 123, 378–390. Krueger, D. D., Osterweil, E. K., Chen, S. P. et al. (2011) Cognitive dysfunc- Hagerman, R., Hoem, G. and Hagerman, P. (2010) Fragile X and autism: tion and prefrontal synaptic abnormalities in a mouse model of Intertwined at the molecular level leading to targeted treatments, fragile X syndrome, Proceedings of the National Academy of Molecular Autism, 1, 12. Sciences of the USA, 108, 2587–2592. Harrison, C. J., Jack, E. M., Allen, T. D. et al. (1983) The fragile X: a scanning Laggerbauer, B., Ostareck, D., Keidel, E. M. et  al. (2001) Evidence that electron microscope study, Journal of Medical Genetics, 20, fragile X mental retardation protein is a negative regulator of trans- 280–285. lation, Human Molecular Genetics, 10, 329–338. Henry, S. A., Lehmann-Masten, V., Gasparini, F. et al. (2002) The mGluR5 Lea, P. M., Movsesyan, V. A. and Faden, A. I. (2005) Neuroprotective activ- antagonist MPEP, but not the mGluR2/3 agonist LY314582, aug- ity of the mGluR5 antagonists MPEP and MTEP against acute excito- ments PCP effects on prepulse inhibition and locomotor activity, toxicity differs and does not reflect actions at mGluR5 receptors, Neuropharmacology, 43, 1199–1209. British Journal of Pharmacology, 145, 527–534. Huber, K. M., Kayser, M. S. and Bear, M. F. (2000) Role for rapid dendritic Levenga, J., Hayashi, S., de Vrij, F. M. S. et  al. (2011) AFQ056, a new protein synthesis in hippocampal mGluR-dependent long-term mGluR5 antagonist for treatment of fragile X syndrome, depression, Science, 288, 1254–1256. Neurobiology of Disease, 42, 311–317. Huber, K. M., Roder, J. C. and Bear, M. F. (2001) Chemical Induction of Li, J., Pelletier, M. R., Perez Velazquez, J. L. et al. (2002) Reduced cortical mGluR5- and protein synthesis-dependent long-term depres- synaptic plasticity and GluR1 expression associated with fragile X sion in hippocampal area CA1, Journal of Neurophysiology, 86, mental retardation protein deficiency, Molecular and Cellular 321–325. Neuroscience, 19, 138–151. Huber, K. M., Gallagher, S. M., Warren, S. T. et al. (2002) Altered synaptic plas- Loesch, D. Z., Huggins, R. M., Bui, Q. M. et al. (2003) Relationship of defi- ticity in a mouse model of fragile X mental retardation, Proceedings of cits of FMR1 gene specific protein with physical phenotype of fragile the National Academy of Sciences of the USA, 99, 7746–7750. X males and females in pedigrees: a new perspective, American Journal of Medical Genetics, 118A, 127–134. Incorpora, G., Sorge, G., Sorge, A. et  al. (2002) Epilepsy in fragile X syndrome, Brain and Development, 24, 766–769. Lu, Y. M., Jia, Z., Janus, C. et al. (1997) Mice lacking metabotropic gluta- mate receptor 5 show impaired learning and reduced CA1 long- Irwin, S. A., Galvez, R. and Greenough, W. T. (2000) Dendritic spine struc- term potentiation (LTP) but normal CA3 LTP, The Journal of tural anomalies in fragile-X mental retardation syndrome, Cerebral Neuroscience, 17, 5196–5205. Cortex, 10, 1038–1044. Manahan-Vaughan, D., Kulla, A. and Frey, J. U. (2000) Requirement of Jacquemont, S., Curie, A., Des Portes, V. et al. (2011) Epigenetic modifica- translation but not transcription for the maintenance of long-term tion of the FMR1 gene in fragile X syndrome is associated with depression in the CA1 region of freely moving rats, The Journal of differential response to the mGluR5 antagonist AFQ056, Sci Transl Neuroscience, 20, 8572–8576. Med, 64, 64ra1. Johnson, B. G., Wright, R. A., Arnold, M. B. et al. (1999) [3H]-LY341495 as a Martin, J. P. and Bell, J. (1943) A pedigree of mental defect showing novel antagonist radioligand for group II metabotropic glutamate sex-linkage, Journal of Neurology, Neurosurgery and Psychiatry, 6, (mGlu) receptors: characterization of binding to membranes of mGlu 154–157. 10 Bioscience Horizons • Volume 6 2013 Review article McBride, S. M. J., Choi, C. H., Wang, Y. et al. (2005) Pharmacological res- Sherman, S. L., Jacobs, P. A., Morton, N. E. et al. (1985) Further segrega- cue of synaptic plasticity, courtship behavior, and mushroom body tion analysis of the fragile X syndrome with special reference to defects in a Drosophila model of fragile X syndrome, Neuron, 45, transmitting males, Human Genetics, 69, 289–299. 753–764. Snyder, E. M., Philpot, B. D., Huber, K. M. et  al. (2001) Internalization of McLennan, Y., Polussa, J., Tassone, F. et  al. (2011) Fragile X syndrome, ionotropic glutamate receptors in response to mGluR activation, Current Genomics, 12, 216–224. Nature Neuroscience, 4, 1079–1085. Merenstein, S. A., Sobesky, W. E., Taylor, A. K. et  al. (1996) Molecular- Vanderklish, P. W. and Edelman, G. M. (2002) Dendritic spines elongate clinical correlations in males with an expanded FMR1 mutation, after stimulation of group 1 metabotropic glutamate receptors in American Journal of Medical Genetics, 64, 388–394. cultured hippocampal neurons, Proceedings of the National Academy of Sciences of the USA, 99, 1639–1644. Musumeci, S. A., Bosco, P., Calabrese, G. et al. (2000) Audiogenic seizures susceptibility in transgenic mice with fragile X syndrome, Epilepsia, Verkerk, A. J., Pieretti, M., Sutcliffe, J. S. et  al. (1991) Identification of a 41, 19–23. gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome, Cell, Oliet, S. H., Malenka, R. C. and Nicoll, R. A. (1997) Two distinct forms of 65, 905–914. long-term depression coexist in CA1 hippocampal pyramidal cells, Neuron, 18, 969–982. Weiler, I. J., Irwin, S. A., Klintsova, A. Y. et al. (1997) Fragile X mental retar- dation protein is translated near synapses in response to neu- Pieretti, M., Zhang, F. P., Fu, Y. H. et al. (1991) Absence of expression of the rotransmitter activation, Proceedings of the National Academy of FMR-1 gene in fragile X syndrome, Cell, 66, 817–822. Sciences of the USA, 94, 5395–5400. Pouladi, M. (2011) Fragile X syndrome therapy: to respond or not to respond Wilson, B. M. and Cox, C. L. (2007) Absence of metabotropic glutamate may be a matter of methylation, Clinical Genetics, 79, 508–510. receptor-Mediated plasticity in the neocortex of fragile X mice, Proceedings of the National Academy of Sciences of the USA, 104, Reddy, K. S. (2005) Cytogenetic abnormalities and fragile-x syndrome in 2454–2459. Autism Spectrum Disorder, BMC Med Genet, 6, 3. Yan, Q. J., Rammal, M., Tranfaglia, M. et  al. (2005) Suppression of two Sandler, A. (2005) Placebo effects in developmental disabilities: major fragile X syndrome mouse model phenotypes by the mGluR5 implications for research and practice, Mental Retardation antagonist MPEP, Neuropharmacology, 49, 1053–1066. and Developmental Disabililities Research Reviews, 11, 164–170. Zou, D., Huang, J., Wu, X. et al. (2007) Metabotropic glutamate subtype 5 Sherman, S. L., Morton, N. E., Jacobs, P. A. et  al. (1984) The marker (X) receptors modulate fear-conditioning induced enhancement of syndrome: a cytogenetic and genetic analysis, Annals of Human prepulse inhibition in rats, Neuropharmacology, 52, 476–486. Geneticsf, 48, 21–37.

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Bioscience HorizonsOxford University Press

Published: Feb 11, 2013

Keywords: fragile X syndrome mGlu5 mGlu theory Fmr1 FMRP

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