4.1. Fragile X mental retardation protein (FMRP)
The first clinical case of fragile X syndrome was described by James Purdon Martin and Julia Bell in 1943 (Martin and Bell, 1943), but it was not until 1991 that the fragile X mental retardation 1 (fmr1) gene was discovered (Verkerk et al., 1991). Indeed, Fragile X syndrome (FXS) is the most frequent inherited form of human mental retardation, with approximately one in 4000 males and one in 8000 females affected (Turner et al., 1996, Garber et al., 2006). This syndrome is most commonly caused by a triplet repeat expansion (CGG) mutation in the fmr1 gene that is located on the long arm of the X chromosome (xq 27.3)
(Fu et al., 1991, Oberle et al., 1991, Verkerk et al., 1991).
fmr1 polymorphic CGG expansion within the 5´ untranslated region (UTR) of the gene can cause at least two clinically distinct neurological disorders: fragile X-syndrome (FXS), which entails a full mutation (>200 repeats), and fragile X-associated tremor/ataxia syndrome (FXTAS), which entails a premutation (55–200 repeats) (Figure 1-1). In FXS patients, the full-mutation allele leads to hypermethylation (Sutcliffe et al., 1992) and deacetylation (Coffee et al., 1999) of fmr1, which results in the silencing of fmr1 transcription and the absence of the gene product (fragile X mental retardation protein, FMRP) (see Fig 1-1). Although premutation carriers are not at risk for fragile X syndrome, it is an important cause of motor syndromes (tremor or ataxia) in aging FXTAS men (Hagerman and Hagerman, 2004, Baba and Uitti, 2005).
FXS is an X-linked inheritable human disease; thus, males are typically more severely affected than females. The clinical features of Fragile X syndrome include physical, developmental and behavioral characteristics. The most prominent physical features of FXS include macroorchidism, an elongated face and a high arched palate that are present in adulthood (Hagerman, 2006, Reiss and Hall, 2007). Clinical studies have also revealed that children with fragile X syndrome have behavioral and emotional problems, such as learning disabilities, attention deficit disorder, hyperactivity disorder, anxiety disorder, aggressiveness, hand flapping, and hand biting. Thus, FXS has shown a strong correlation with Autism Spectrum Disorders (ASDs) (Brown et al., 1982, Budimirovic and Kaufmann, 2011). Indeed fmr1
mutation is the most common genetic cause of ASDs, accounting for approximately 5% of cases (Schaefer and Mendelsohn, 2008).
FXS is caused by loss of the fmr1 gene product; the fragile X mental retardation protein (FMRP) is a cytoplasmic mRNA-binding protein that is widely expressed in various tissues with the most abundant expression in the brain and testis (Devys et al., 1993, Hinds et al., 1993). In the brain, the protein is expressed in neurons, particularly those of the hippocampus, amygdala, and in the Purkinje cells of the cerebellum (Abitbol et al., 1993, Devys et al., 1993). FMRP has two KH domains and an RGG domain (RGG box). These domains have been shown to be involved in mRNA binding (Ashley et al., 1993, Siomi et al., 1993a, Siomi et al., 1993b).
Amino acid sequence alignment of FMRP from humans, mice, frogs, zebrafish and fruitflies has revealed high conservation in functional domains, including the nuclear localization signal (NLS), two KH domains, the nuclear export signal (NES) and an RGG box (Figure 1-2).
However, no CGG repeats have been found in the 5′ UTR sequences of zebrafish, frog or fruit-fly fmr1 gene mRNA (van 't Padje et al., 2005).
FMRP plays important roles in the regulation of dendritic mRNA localization and/or synaptic protein synthesis (Feng et al., 1997, Darnell et al., 2001, Zhang et al., 2001, Bassell and Warren, 2008, Kelleher and Bear, 2008). In some case, FXS patients have severe clinical phenotypes that are not caused by the absence of FMRP; rather, they result from mutation in the KH domain. In addition, FMRP has also recently been linked to the microRNA pathway, an important pathway of non-coding small RNA sequences (21–23 nucleotides) that regulates gene
expression (Lee et al., 1993) by modulating associations with Dicer (Cheever and Ceman, 2009). Therefore, recent studies have suggested that dysregulation or loss/dysfunction of FMRP is the cause of FXS-like symptoms.
4.2. The animal models of Fragile X syndrome 4.2.1. The Drosophila model
Previous studies have shown the Drosophila FXS model to be an excellent and simple model system for studying the genetics and molecular aspects of human FXS. Null mutation and overexpression of Drosophila fmr1 (dfmr1) have been widely used to create Drosophila models of FXS (Zhang et al., 2001, Pan et al., 2004). Based on molecular and genetic studies, FMRP function is conserved between Drosophila and humans. For example, Zhang et al. (2001) reported that dFMRP acts as a negative regulator of the microtubule-binding MAP1B homolog Futsch to control synaptic structure and function, suggesting that FMRP acts as a translation repressor in the brain (Zhang et al., 2001). In addition, loss of dFMRP function induced defects in neuronal architecture, disrupted circadian rhythms, impaired social interaction and produced deficits in learning and memory (Dockendorff et al., 2002, Inoue et al., 2002, Morales et al., 2002). The mGluR theory of FXS is the most common mechanistic explanation of the pathophysiology of FXS, as feeding dfmr1 mutants MPEP or class II/III mGluR antagonists can rescue synaptic plasticity, courtship behavior, and mushroom body defects in a Drosophila model of FXS (McBride et al., 2005). Thus, exploring the
strong evolutionary link between mGluR5 receptors and FMRP signaling in Drosophila may be a useful model for testing the efficacy of therapeutic strategies in FXS.
4.2.2. The mouse model
fmr1 is highly conserved between humans and mice with a coding DNA and amino acid sequence identity of 95% and 97%, respectively;
this sequence identity includes the CGG repeat in the 5’ UTR (Ashley et al., 1993). Microscopic analyses of brain material from both FXS patients and fmr1 KO mice have shown dendritic spine abnormalities that include increased spine density and immature spine morphologies in the hippocampus, neocortex, and cerebellum (Hinton et al., 1991, Comery et al., 1997, Nimchinsky et al., 2001, Grossman et al., 2006), suggesting that the loss of FMRP function leads to alterations in dendritic spine structure and deficits of synaptic connectivity and plasticity. fmr1 KO mice exhibit a phenotype with some similarities to humans, such as seizures, macroorchidism and behavioral abnormalities. In behavioral tests, mutant animals displayed hyperactivity, reduced anxiety, reduced social interaction, and learning impairment (Helm et al., 1994, Liu et al., 2011).
Consistent with these behavioral deficits, electrophysiological studies have reported the loss of LTP in the anterior cingulate cortex (ACC) and the lateral amygdala (Zhao et al., 2005) and enhanced group I metabotropic glutamate receptor (mGluR)-dependent LTD in the hippocampus (Huber et al., 2002) and cerebellum (Koekkoek et al., 2005) in fmr1 KO mice.
4.2.3. The zebrafish model
The zebrafish (Danio rerio) is a small tropical freshwater teleost that was first used as a genetic model system in the early 1980s. Due to the accumulated genetic knowledge and tools developed for the zebrafish, they are now considered an excellent and relevant model system in studies of human neurological disorder. Orthologs of the human fmr1 gene have been identified in zebrafish. The deduced amino acid sequence of the zebrafish FMRP consists of 569 residues and shares 72% amino acid identity with human. Pioneering studies found that zebrafish FMRP was ubiquitously expressed throughout embryos at 3 h post-fertilization (hpf); however, in 72-hpf embryos, the most abundant expression of FMRP was in the brain. In adult zebrafish, FMRP is a ubiquitously expressed RNA-binding protein with high expression levels in the telencephalon, diencephalon, metencephalon, spinal cord, cerebellum, and testes (van 't Padje et al., 2005). These findings suggest that zFMRP may play an important role in the developing brain.
The zebrafish embryo has been established as a model for fmr1 loss-of-function analysis using a morpholino antisense oligonucleotide to block of FMRP expression (Tucker et al., 2006). These studies suggested that fmr1 and mGluRs have regulatory functions in axonal branching, guidance and fasciculation that have implications for the synaptic morphology component of FXS. However, studies using TILLING (targeted induced local lesions in genomes) to generate fmr1 knockout alleles in zebrafish found that the fish did not display any phenotype; this was in contrast to the morphant reported in a previous study (den Broeder
et al., 2009). Therefore, it remains to be investigated whether the reported morpholino-based fmr1 phenotypes are due to morpholino’s off-target effects.