Cytoplasmic FMRP Interacting Protein
1 (CYFIP1) – A key player in the pathophysiology of the Fragile X Syndrome?
Supervisor: Dr. M.J.H. Kas (Rudolf Magnus Institute, UMC Utrecht)
Karin Legerstee
Neuroscience and CognitionExperimental and Clinical Neuroscience
3052621
Master Thesis (7.5 ECTs)
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Contents
Page
Abstract
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1: The Fragile X Syndrome
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2: Animal models of FXS
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3: The ‘mGluR theory of fragile X’
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4: Alleviation of FXS (phenotypes) using group 1 metabotropic glutamate receptor antagonists 13
5: The potential role of Cyfip1 in the pathophysiology of Fragile X Syndrome
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Conclusion
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Reference List
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Abstract
This literature study investigates the role Cyfip1 could play in the pathophysiology of the Fragile X
Syndrome (FXS). FXS, the most common form of mental retardation, is caused by disruptions to the
Fmr1 gene. In all cases except one FXS is caused by a loss of expression of the protein encoded for by
Fmr1, FMRP, most commonly due to a CGG-repeat expansion resulting in excessive methylation of
FMRP. In the single exception a point mutation in the FMR1 gene specifically impairs the ability of
FMRP to associate with polyribosomes, while leaving its ability to bind (m)RNA molecules in vivo
intact (De Boulle et al., 1993; Feng et al., 1997a; Siomi et al.,1994). It results in an especially severe
form of FXS. Suggesting that, although FMRP has a number of different functions in cells it is its role
in the regulation of (local) mRNA translation as a binding partner of polyribosomes, mainly repressing
the translation of its target mRNAs, that is crucial to the pathophysiology of FXS (Laggerbauer et al.,
2001; Lu et al., 2004).
As a mechanistic explanation of the pathophysiology of FXS the ‘mGluR theory of fragile X’ is most
commonly accepted (Bear et al., 2004).It also points to the role of FMRP in the regulation of local
mRNA translation as critical for the pathophysiology of FXS. According to this theory the activation of
group 1 metabotropic glutamate receptors (mGluRs) at dendrites stimulates the local translation of
mRNAs that are responsible for the functional effects of mGluR-signalling as well as of FMRP. The
FMRP inhibits the further local translation of these mRNAs, serving as a form of end-product
inhibition for the mGluR-signalling. In the absence of FMRP, as in FXS, the inhibition by FMRP falls
away leading to an exaggeration of all local protein synthesis-dependent effects of mGluR-signalling,
proposed to be the underlying cause of the diverse symptoms of FXS.
The Fmr1 knockout mouse model of FXS displays a multifaceted phenotype that is generally consist
with the symptoms seen in FXS patients and includes phenotypes analogous to the physical,
neurological, structural and behavioural characteristics of FXS in humans (Bakker et al., 1994).
However, the hallmark mental retardation appears to be more effectively replicated in the dFmr1
loss of function Drosophila FXS model (Bolduc et al., 2008; Choi et al., 2010; McBride et al., 2005;
Wan et al., 2000). In support of the ‘mGluR theory of FXS’ it has been demonstrated that several local
protein synthesis-dependent effects of mGluR-signalling are enhanced in a range of different brain
structures in animal models of FXS. Through these effects the theory can be envisioned to explain
most symptoms of FXS. Most convincingly, the relevance of excessive group1 mGluR-signalling to FXS
symptoms is directly demonstrated by the significant rescue of neurological, physical and cellular
aspects of FXS in mice with complete knockout of Fmr1 as well as a 50% knockdown of Grm5, the
gene coding for the group 1 metabotropic glutamate receptor mGluR5 (Dolen et al., 2007). Similarly,
pharmacological inhibition of group 1 mGluR-signalling in the Fmr1 knockout mouse significantly
rescues phenotypes analogous to neurological, cellular and behavioural characteristics of FXS
(Chuang et al., 2005; de Vrij et al., 2008; Levenga et al., 2011; Su et al., 2011; Yan et al., 2005). In the
Drosophila model inhibiting group 1 mGluR signalling was also able to rescue the cognitive deficits
seen in these flies (Bolduc et al., 2008; Choi et al., 2010; McBride et al., 2005). Establishing the
validity of the main points of the ‘mGluR theory of FXS’, including the assumption that the role of
FMRP in local protein synthesis is crucial for the pathophysiology of FXS.
Cyfip1 is a known cytoplasmic interactor of FMRP (Schenck et al., 2001). Genetic studies have
provided some data suggesting that Cyfip1 might play a role in some of the symptoms of FXS,
especially its autistic features (Butler et al., 2004; Doornbos et al., 2009; Milner et al., 2005; Murthy
et al., 2007; Nishimura et al., 2007; Nowicki et al., 2007; van der Zwaag et al., 2010). However, it is
the finding that Cyfip1 as an eukaryotic initiation factor 4E Binding Protein (4E-BP) mediates the
inhibition of local mRNA translation by FMRP, that convincingly points to a crucial role of Cyfip1 in
the pathophysiology of FXS (Napoli et al., 2008). For many of the FMRP-target mRNAs, which would
be expected to include mRNAs crucial for many of the local protein-synthesis dependent effects of
mGluR-signalling, it is in fact Cyfip1 that is mediating the effects of FMRP on local mRNA translation.
Placing CYFIP1, just like FMRP, at the heart of the ‘mGluR theory of FXS’ and warranting a much more
extensive investigation into the role of Cyfip1 in the pathophysiology of FXS.
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Chapter 1: The Fragile X Syndrome
Fragile X Syndrome (FXS) is the most common form of heritable mental retardation, it affects about
one in 4000 males and one in 8000 females (Jin and Warren, 2000). Apart from mental retardation,
FXS is characterised by a long and relatively narrow face and large protruding ears (Loesch et al.,
2003). Children with FXS also show accelerated pre-pubescent growth and decreased body weight
(Loesch et al., 1995; Loesch et al., 2003). Neuroimaging studies have shown selective changes in the
brain size of fragile X patients, as well as differences in the white matter (Barnea-Goraly et al., 2003).
One of the most common clinical features of FXS is heightened sensitivity to sensory stimulation and
increased cortical auditory evoked potentials have been measured in FXS patients (Castren et al.,
2003; Frankland et al., 2004; Miller et al., 1999). The neurological phenotype of FXS includes
childhood epilepsy (Musumeci et al., 1999). On a structural level, FXS is characterised by spine
abnormalities (Irwin et al., 2000; Irwin et al., 2001). In fragile X patients long thin spines with an
immature morphology are significantly more abundant and thicker spines with a more mature
morphology are significantly less abundant than in healthy controls. It has long been hypothesised
that there is a link between spine abnormalities and mental retardation in humans (Purpura, 1974).
In at least 98% of Fragile X patients the cause of FXS is a lack of Fragile X Mental Retardation Protein
(FMRP, the product of the X-linked FMR1 gene) expression, due to expansion of a CGG repeat in the
5’-untranslated region of FMR1 (Fu et al., 1991; Oberle et al., 1991; Sutcliffe et al., 1992; Verkerk et
al., 1991). When the extension exceeds around 200 trinucleotide repeats excessive methylation of
the repeat and the promoter region ensues, resulting in transcriptional silencing of the gene. In the
remaining 2% of patients FXS is also caused by deficient FMRP expression due to several different
other mutations (Hirst et al., 1995; Lugenbeel et al., 1995; Meijer et al., 1994). There is only a single
exception where patients have normal expression of FMRP but still show an especially severe form of
FXS (De Boulle et al., 1993). In this case the cause of FXS is a single point mutation leading to an
isoleucine to asparagine substitution in the second KH domain of FMRP (Siomi et al., 1994).
As a single protein strongly linked to a specific form of mental retardation FMRP has been well
studied, not only in the hope of alleviating FXS through medication, but also because of the
possibility of gaining some insight into the molecular mechanisms of cognition. Because it is so well
studied, quite a lot is known about FMRP. FMRP is an RNA-binding protein known to interact with
several different RNA-molecules, mostly messenger RNA (mRNA) and ribosomal RNA (rRNA)
molecules (Antar et al., 2005; Brown et al., 2001; Miyashiro et al., 2003). FMRP contains several
different RNA-binding domains including a RGG box at the C-terminus and two KH domains located
roughly in the middle of the FMRP protein (Bardoni and Mandel, 2002). In some instances FMRP
recognises the RNA molecules directly, in other cases the interaction occurs through small noncoding RNA adaptors such as the brain cytoplasmic RNA 1 (BC1) (Bagni and Greenough, 2005). FMRP
is widely expressed throughout development as well as in adulthood, but it is most strongly
expressed by Neurons (Devys et al., 1993). In all expressing cells at any given time the vast majority
of FMRP is found in the cytoplasm, despite the fact that it possesses both nuclear localisation (NLS)
and nuclear export (NES) signals (Devys et al., 1993; Feng et al., 1997b; Fridell et al., 1996; Sittler et
al., 1996). In neurons FMRP is found in neurites in the form of granules, at synapses and in growth
cones (Antar et al., 2004; Antar et al., 2005). FMRP is associated with actively translating
polyribosomes as a part of messenger ribonucleoprotein (mRNP) particles, suggesting a role in the
regulation of (local) mRNA translation (Feng et al., 1997a; Feng et al., 1997b). Indeed, FMRP has been
shown to influence the translation of its target mRNAs, mostly it acts a translation repressor
(Laggerbauer et al., 2001; Lu et al., 2004). Apart from a more direct role as a binding partner of
polyribosomes, FMRP may also regulate the translation of its target mRNAs through RNA
interference as it is a component of the RNA-Induced Silencing Complex (RISC) (Caudy et al., 2002;
Ishizuka et al., 2002; Jin et al., 2004). Furthermore FMRP can act as an kinesin adaptor and plays an
important role in mRNA transport, for a review see Bassel et al 2008 (Bassell and Warren, 2008). The
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transport of FMRP to dendrites appears to be through active transport of kinesin along microtubules
as an mRNP particle and is induced by activation of the group1 metabotropic glutamate receptor 5
(mGluR5) (Antar et al., 2004; Antar et al., 2005; De Diego Otero et al., 2002; Dictenberg et al., 2008).
To the pathology of FXS however, it seems the role of FMRP as binding partner of polyribosomes is
most important. An isoleucine to asparagine mutation in the second KH domain of FMRP is the
genetic mutation underlying the single case in which FXS patients have normal expression of FMRP, it
causes an especially severe form of FXS (De Boulle et al., 1993; Siomi et al., 1994). Unexpectedly,
considering the KH domain is an RNA-binding domain, the ability of the mutated FMRP to bind mRNA
molecules in vivo is entirely unaffected (Feng et al., 1997b). However the ability of the mutated
FMRP to associate with polyribosomes is severely impaired. In patient cells more than 90% of the
mutated FMRP is present as free protein, whereas there is rarely any free FMRP protein detected in
healthy control cells where it is instead found in the polyribosome fraction. This specific impairment
of FMRP-polyribosome interactions but not of FMRP-(m)RNA interactions in an especially severe FXS
type indicates FMRP-polyribosome interactions are essential for FXS. This strongly suggests the
absence of the translational regulation of FMRP through its direct interaction with polyribosomes is
crucial for the pathophysiology of FXS, rather than aberration of mRNA transport by FMRP or
impairment of translational inhibition through its association with the RISC complex.
This literature study investigates the potential role of Cytoplasmic FMRP Interacting Protein 1
(Cyfip1) in the pathophysiology of FXS. To study FXS two different animal models are commonly
used, a mouse Fmr1 knockout model and a Drosophila dFMR1 loss of function mutant. The
phenotype displayed by these animals, which is multifaceted and generally consistent with the
human symptoms of FXS, will be briefly presented here to demonstrate the validity of using these
animal models in the study of FXS. This is followed by a review of the large amount of data obtained
by the study of these animal models that supports the most commonly accepted mechanistic
explanation of the pathophysiology of FXS, the ‘mGluR theory of Fragile X’ (Bear et al., 2004). Finally
the data currently available in the literature hinting at a role for Cyfip1 in FXS will be discussed,
followed by a detailing of the crucial role Cyfip1 plays in the repression of local mRNA translation by
FMRP and why this places Cyfip1 at the heart of the ‘mGluR theory of fragile X’.
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Chapter 2: Animal models of FXS
As a form of mental retardation with such high prevalence and caused by disruptions to one single
gene FXS forms an ideal mental disorder to study using animal models. In 1994 the Dutch Belgian
Consortium successfully generated Fmr1 knockout mice with a C57BL/J6 background (Bakker et al.,
1994). The FMR1 gene is highly conserved amongst all species and the amino acid sequence of the
mouse and human FMR1 gene show 97% homology (Ashley et al., 1993; Verkerk et al., 1991). The
mouse fmr1 gene, like the human FMR1 gene, is located on the X-chromosome and also incorporates
CGG repeats in its promoter region. Furthermore, the expression patterns and expression levels of
FMR1 in mice and humans are very similar, both at the mRNA as well as at the protein level (Abitbol
et al., 1993; Bachner et al., 1993; Bakker et al., 2000; Devys et al., 1993; Hinds et al., 1993). Together
this makes the Fmr1 knockout mouse a potentially very valid animal model to study FXS. And indeed,
the Fmr1 knockout mouse displays a multifaceted phenotype that is generally consistent with the
symptoms seen in human fragile X patients.
Phenotypes seen in the Fmr1 knockout mouse that can be linked to FXS symptoms include some
physical features. Macroorchidism is one of the key physical features of FXS, progressive
enlargement of the testes after puberty is observed in the vast majority of male FXS patients (de
Vries et al., 1998; Thake et al., 1985; Turner et al., 1980). Fmr1 knockout mice also show progressive
macroorchidism after puberty, the weight increase of the testes in knockout mice becomes gradually
more significant over time, while there is no difference in the total weight or in the weight of a range
of different organs between knockout mice and wild type (WT) mice (Bakker et al., 1994; Dolen et al.,
2007; Slegtenhorst-Eegdeman et al., 1998). Another physical feature of the Fragile X Syndrome is
increased prepubescent growth in stature (Loesch et al., 1995). Since FXS patients show decreased
pubescent growth, their height normalises after puberty. This same growth pattern is also seen in
FMR1 knockout mice, with increased prepubescent growth in Fmr1 knockout mice compared to WT
mice (significant from postnatal day 26 onwards) and the growth increase no longer being apparent
after puberty (postnatal day 45) (Dolen et al., 2007).
The neurological phenotype of FXS includes seizures. The prevalence of epilepsy in FXS patients is 1020% (Berry-Kravis, 2002; Incorpora et al., 2002; Musumeci et al., 1999; Wisniewski et al., 1991).
Mainly childhood epilepsy is seen, with the majority of patients growing out of their seizures by the
end of adolescence. Next to seizures, roughly 50% of prepuberal FXS patients show abnormalities in
their EEG readings, namely epileptiform spikes. Analogous abnormalities have been reported for
Fmr1 knockout mice, hippocampal slices of Fmr1 knockout mice show prolonged epileptiform
discharges in response to synaptically released glutamate, which are inhibited by translational
inhibitors (Chuang et al., 2005).
However, spontaneous seizure activity has not been reported for Fmr1 knockout mice. On the other
hand, Fmr1 knockout mice have a markedly enhanced sensitivity to audiogenic seizures (Chen and
Toth, 2001; Dolen et al., 2007; Yan et al., 2004). The genetic background has been shown to have
some effect on this phenotype, with significantly enhanced sensitivity seen in WT mice of some
inbred strains compared to WT F1 hybrids (Errijgers et al., 2008; Yan et al., 2005). However, Fmr1
knockout mice of these inbred strains are still significantly more sensitive to audiogenic seizures than
WT inbred mice, as are the hybrid Fmr1 knockout mice although they are not as sensitive to the
seizures as the inbred Fmr1 knockout mice. Furthermore, two separate studies using mice of several
different genetic backgrounds used an audiogenic seizure paradigm that resulted in audiogenic
seizures in about 60-70% of Fmr1 knockout mice but did not cause any seizures in WT mice of the
same genetic background (Chen and Toth, 2001; Dolen et al., 2007). Indicating the enhanced
sensitivity seen in Fmr1 knockout mice is due to the loss of FMRP and thus a true phenotype of the
Fmr1 knockout mice. In one study the enhanced sensitivity of Fmr1 knockout mice to audiogenic
seizures was confirmed by using immunohistochemistry to determine the expression of c-Fos, an
immediate-early gene product whose expression is induced in neurons by epileptic seizures. Fmr1
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knockout mice showed high expression of c-Fos following exposure to the audiogenic seizure
paradigm, whereas no c-Fos was detected in exposed WT mice of the same genetic background
(Chen and Toth, 2001). Furthermore, in some genetic backgrounds the Fmr1 knockout mice loose the
sensitivity to audiogenic seizures before adulthood, consistent the childhood epilepsy seen in FXS.
Thus, enhanced sensitivity to audiogenic seizures is a robust phenotype seen in Fmr1 knockout mice
of a variety of different genetic backgrounds, indicating the enhanced sensitivity is a result of the loss
of FMRP in the knockout mice. It mimics the phenotype of mainly childhood epilepsy seen in FXS
patients, although the Fmr1 knockout mice do not show spontaneous seizure activity and their
enhanced sensitivity to seizure activity seems to be specific for the auditory system, as they do not
show enhanced sensitivity to a range of different chemical convulsants (Chen and Toth, 2001).
In fact, the enhanced sensitivity to audiogenic seizures can also be viewed as an extension of another
symptom of FXS, namely sensory hyperexcitability. Clinically it has been observed that sensory
stimulation of FXS patients can result in such symptoms as hyperarousal, hyperactivity and autisticlike responses (Hagerman et al., 2009). Hyperreactivity of FXS patients to sensory stimuli has also
been demonstrated more directly. FXS patients show an increased magnitude of electrodermal
responses to a wide range of sensory stimuli, as well as an increased number of electrodermal
responses per stimulation and lower rates of habituation (Miller et al., 1999). FXS patients also
display a shorter interbeat interval in their heart rate in response to sensory stimulation (Boccia and
Roberts, 2000). Auditory stimulation results in a larger amplitude of the cortical auditory evoked
potentials measured by EEG in FXS patients, and habituation of this response is significantly
decreased in the FXS patients (Castren et al., 2003). Lastly, using eye tracking technology, the
pupillary activity of FXS patients was shown to be significantly enhanced in response to visual stimuli
(photos of faces) (Farzin et al., 2009).
Another FXS symptom related to enhanced sensitivity to sensory stimulation is the deficit in prepulse
inhibition (PPI) of the acoustic startle response seen in FXS patients. In this paradigm a low intensity
auditory prepulse decreases the subsequent response to a loud startling noise, and it is used to
investigate basic sensorimotor processing. In normally developing age-matched control subjects the
startle response, measured through EMG responses from the lower eyelid, is substantially reduced if
it is preceded by the prepulse, but PPI is markedly reduced in FXS patients (Frankland et al., 2004;
Hessl et al., 2009). In Fmr1 knockout mice several early studies seemed to suggest PPI is increased in
Fmr1 knockout mice on most genetic backgrounds (Frankland et al., 2004; Nielsen et al., 2002; Paylor
et al., 2008). However, for these studies a relatively insensitive measure for the acoustic startle
response, whole body startle response, was used. Recent studies using much more sensitive methods
to measure startle responses, namely eyelid responses analogous to the method used in the human
studies, found a marked deficit in the PPI of the acoustic startle response in Fmr1 knockout mice (de
Vrij et al., 2008; Levenga et al., 2011).
Behavioural symptoms of FXS include hyperactivity (Hagerman et al., 2009). Fmr1 knockout mice
with a very diverse set of different genetic backgrounds have been shown to behave significantly
differently from WT mice in the open field, with differences on a range of different parameters all
suggesting hyperactivity and increased exploratory behaviour of Fmr1 knockout mice (de DiegoOtero et al., 2009; Liu et al., 2011; Min et al., 2009; Peier et al., 2000; Qin et al., 2002; Yan et al.,
2004; Yan et al., 2005).
However, of course the most important behavioural symptom of FXS is the mental retardation and
the learning disability. Unfortunately, this particular symptom does not seem to be replicated as
strongly in the Fmr1 knockout mouse. Fmr1 knockout mice do show some cognitive impairments, but
overall the cognitive abilities of the knockout mouse do not appear to be affected as strongly as in
FXS patients (Bakker and Oostra, 2003). Recent studies suggest the higher cognitive functions, such
as attention, cognitive flexibility and inhibitory control, might be affected a bit more severely in the
Fmr1 knockout mice, more analogous to the cognitive problems seen in FXS patients (Casten et al.,
2011; Krueger et al., 2011; Moon et al., 2006). More robust cognitive phenotypes seen in Fmr1
knockouts also include learning deficits seen in both the passive avoidance task and an olfactory
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discrimination task, as well as the inability to discriminate between a novel object and a familiar one
(Larson et al., 2008; Liu et al., 2011; Ventura et al., 2004).
Another key feature of FXS are the alterations in dendritic spines, first reported by Rudelli et
al(Rudelli et al., 1985). An increase in spines of the ‘immature’ type, long thin and tortuous, with a
concomitant decrease in ‘mature’ short and thick mushroom spines has been reported, as well as a
higher spine density at the basal branches for layer V pyramidal cell of the cerebral cortex of FXS
patients (Irwin et al., 2000; Irwin et al., 2001). The same dendritic spine phenotype is also seen in the
cerebral cortex of Fmr1 knockout mice (Comery et al., 1997; Dolen et al., 2007; Irwin et al., 2002). For
pyramidal neurons in the hippocampus region specific dendrite abnormalities have also been
reported (Levenga et al., 2011). In the CA1 region of the hippocampus the pyramidal cells of Fmr1
knockout mice have significantly more long thin tortuous spines as well as an increased spine density
compared to WT mice, yet in the CA3 region there was no significant difference between knockout
and WT mice.
A final interesting phenotype seen in the Fmr1 knockout mice is the enhanced ocular dominance
plasticity (Dolen et al., 2007). The ocular dominance paradigm is used to investigate experiencedependent plasticity of the brain, where temporary monocular deprivation initially causes depression
of the deprived eye followed by potentiation of the non-deprived eye. The Fmr1 knockouts show
normal depression of the deprived eye, but significantly increased potentiation of the non-deprived
eye.
The other important animal model for fragile is the knockout of the Drosophila homologue of FMRP,
dFMR1 (Wan et al., 2000). Due to the fact that this is a Drosophila-based animal model, it is harder to
link the phenotypes seen in this animal model to human fragile X symptoms than it is for FMR1
knockout mice, but some parallels can be found. The dfmr1 knockout Drosophila show courtship
behaviour deficits as well as mushroom body deficits, phenotypes not easily linked to the human
disorder, but they also display cognitive deficits in the experience-dependent modification of
courtship behaviour (Dockendorff et al., 2002; McBride et al., 2005). Namely, in a conditioned
courtship paradigm they show normal learning but fail to display any memory of their training, both
in immediate recall tests as well as in short-term memory tests. Additionally, dFMR1 knockout flies
display deficits in olfactory learning and in long-term olfactory memory (Bolduc et al., 2008). Lastly,
dFmr1 knockout mice have been shown to display significantly enhanced age-related cognitive
decline, namely an age-dependent loss of learning during training (Choi et al., 2010). Such cognitive
deficits can of course be linked to the cognitive impairments seen in Fragile X patients, probably the
most significant feature of the fragile X syndrome and a symptom which has proved hard to
recapitulate in the Fmr1 knockout mouse model.
Other phenotypes that may be linked to the human disorder include disruption of the circadian
rhythms (Dockendorff et al., 2002; McBride et al., 2005; Morales et al., 2002). Additionally, dFMR1
loss of function mutants as well as dFmr1 knockouts show dendritic spine abnormalities (Lee et al.,
2003; Morales et al., 2002). The dendritic arborisation neurons of Drosophila dFMR1 loss of function
mutants have an increased density of dendrites, recapitulating part of the spine morphology
abnormalities seen in both Fmr1 knockout mice and fragile X patients (Lee et al., 2003). The
drosophila dFMr1 knockout flies show abnormal neurite extension, guidance and branching across a
range of different neuronal cell types (Morales et al., 2002).
Taken together the Drosophila FXS model, like Fmr1 knockout mice, shows a range of different
phenotypes that can be linked to the symptoms of FXS in humans. However, by far the most
important advantage of the Drosophila FXS model over the mouse model is that it efficiently
replicates the key FXS feature of mental retardation.
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Chapter 3: The ‘mGluR theory of fragile X’
As a theory to explain the pathophysiology of fragile X, the ‘mGluR theory of fragile X’ is becoming
gradually more and more widely accepted. According to this theory many of the diverse symptoms of
fragile X syndrome are due to excessive group 1 metabotropic glutamate receptor (mGluR) signalling
(Bear et al., 2004). In the central nervous system there are two classes of receptors for the main
excitatory neurotransmitter glutamate, the fast ligand-gated ionotropic receptors and the slower Gprotein coupled receptors. There are three different types of ionotropic glutamate receptors in the
central nervous system, AMPA receptors, kainate receptors and NMDA receptors. The metabotropic
glutamate receptors are also divided into three groups based on their structure, signal transduction
mechanism and pharmacological properties. Group 2 and 3 mGluRs inhibit adenylyl cyclases through
Gi/o-proteins. The Group 1 mGluRs, implicated in FXS by the ‘mGluR theory of fragile X’, are coupled
to Gq-proteins and stimulate phospholipase C (PLC). PLC synthesises the second messengers inositol
triphosphate (IP3) and diacyl glycerol (DAG) iniating a signalling cascades that includes stimulation of
adenylyl cyclase. In mammals the group 1 mGluRs is made up by mGluR1 and mGluR5. The two
receptors have different tissue and subcellular expression patterns, in the forebrain mGluR5 is the
main postsynaptic group 1 mGluR, in the cerebellum mGluR1 is predominantly expressed. In the
model for the pathophysiology of FXS set forth in the ‘mGluR theory of fragile X’ group 1 mGluR
signalling stimulates the local protein synthesis of proteins mediating the functional effects of
mGluR-signalling as well as the protein synthesis of FMRP. The FMRP then functions to inhibit further
local translation of these proteins, in effect functioning as a form of end-product inhibition. In FXS
the inhibition by FMRP falls away leading to exaggeration of all protein-synthesis dependent
functional consequences of group 1 mGluR-signalling, according to the theory this is the underlying
cause for many of the diverse symptoms of FXS.
As hypothesised in the ‘mgluR theory of fragile X’, group 1 mGluR signalling stimulates FMRP
expression. Stimulation of these receptors rapidly increases the expression of FMRP in
synaptoneurosomes and in primary cortical neurons (Todd et al., 2003a; Weiler et al., 1997). In
hippocampal slices induction of mGluR-dependent LTD rapidly increases the expression of FMRP
(Hou et al., 2006). In vivo whisker stimulation, a method of evoking experience dependent plasticity,
increases the expression of FMRP in the barrel cortex which requires group 1 mGluR signalling (Todd
et al., 2003b). In all these instances the upregulation of FMRP is inhibited by translation inhibitors,
indicating group 1 mGluR signalling stimulates the translation of Fmr1 mRNA into FMRP as suggested
by the ‘mGluR theory of fragile X’. Using immunofluorescence confocal microscopy stimulation of
group 1 mGluR signalling has been shown to upregulate the expression of FMRP, as well as FMRPmRNP complexes, at postsynaptic spines, which is again largely mediated by increased translation of
Fmr1 mRNA (Ferrari et al., 2007)(Todd et al., 2003b).
Since group 1 mGluR signalling stimulates local protein synthesis in dendrites, one of the
consequences of the ‘mGluR theory of FXS’ would be abnormal protein synthesis in FXS. If FMRP
indeed functions inhibit group 1 mGluR signalling, then the loss of FMRP would be expected to result
in elevated basal protein synthesis. Elevated in vivo basal protein synthesis has been demonstrated in
many regions of the cerebral cortex of the Fmr1 knockout mouse (Qin et al., 2005). Basal protein
synthesis levels were especially elevated in the thalamus, the hypothalamus and the hippocampus of
Fmr1 knockout mice as compared to WT mice, but basal protein synthesis was also significantly
elevated in the basolateral amygdala, the mammillary bodies, the raphe nuclei, and the dorsal motor
nucleus of the Vagus. Increased basal protein synthesis was also demonstrated in hippocampal slices
of Fmr1 knockout mice (Dolen et al., 2007). By electrophoresis the elevated rate of basal protein
synthesis in the hippocampus of Fmr1 knockout mice was shown to include proteins of many
different molecular weights, rather than being limited to just a few principal species. Translation of
most of the large number of FMRP-target mRNAs is also constitutively elevated in Fmr1 knockout
mice, on average basal translation of the FMRP-target mRNAs is increased about 2-fold as compared
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to WT mice (Berry-Kravis et al., 2011; Hou et al., 2006). Stimulation of mGluRs and mGluR-dependent
LTD enhances the expression of FMRP-binding mRNAs. Hence increased constitutive protein
synthesis in general and of FMRP-target mRNAs specifically, in FXS as in Fmr1 knockout mice, is an
expected consequence of excessive mGluR signalling and thus these findings support the ‘mGluR
theory of fragile X’.
More counterintuitive is the finding that the activation of protein translation in response to mGluR
stimulation or mGluR-dependent LTD is significantly reduced in Fmr1 knockout compared to WT
mice. In synaptoneurosomes the enhancement of polyribosomal assembly and rate of protein
synthesis in response to depolarisation or to stimulation with the group 1 mGluR agonist DHPG is
significantly reduced in Fmr1 knockout compared to WT mice (Greenough et al., 2001; Weiler et al.,
2004). More specifically, the increase in translation of many of the FMRP-target mRNAs seen in
response to mGluR activation is highly reduced in Fmr1 knockout mice (Berry-Kravis et al., 2011).
Furthermore, in hippocampal slices induction of mGluR-dependent LTD causes a significantly
decreased increase in the translation of FMRP-target mRNAs in Fmr1 knockout compared to WT mice
(Hou et al., 2006). Although perhaps counterintuitive, these findings are also in line with the ‘mGluR
theory of fragile X’. The constitutively elevated protein synthesis seen in Fmr1 knockout mice, in
response to the abnormally high group 1 mGluR-signalling during basal conditions caused by the loss
of inhibition by FMRP, also means the effect of actual activation of the mGluRs on protein synthesis,
by glutamate or synthetic agonists like DHPG, will be reduced since mGluR-signalling was already
elevated under basal conditions. Thus a decreased elevation in the translation of proteins in
response to stimulation of the group 1 mGluRs suggests the loss of FMRP indeed leads to excessive
mGluR signalling supporting the ‘mGluR theory of Fragile X’.
Apart from local protein synthesis group 1 mGluR-signalling has also been shown to stimulate the
internalis(Nakamoto et al., 2007)ation of AMPA receptors. Stimulation of cultured hippocampal
neurons with DHPG has been demonstrated to result in an increase in the internalisation of AMPA
receptors with a corresponding decrease in the expression of AMPA receptors at the surface of
synapses using a variety of different methods (Nakamoto et al., 2007; Snyder et al., 2001; Xiao et al.,
2001)(Snyder et al., 2001). AMPA receptor internalisation in response to DHPG-stimulation depends
on local translation but does not require transcription. In the cortex of Fmr1 knockout mice the
expression of AMPA receptors at synapses is reduced (Li et al., 2002). The same is seen at synapses of
the lateral amygdala in Fmr1 knockout mice (Suvrathan et al., 2010). The knockdown of FMRP by
siRNA in cultured hippocampal neurons in the absence of any exogenously applied agonists causes an
increase in AMPA receptor internalisation and decreased AMPA receptor surface expression at the
postsynaptic membrane (Nakamoto et al., 2007). Furthermore, like for local protein synthesis the
response of AMPA receptor internalisation to group 1 mGluR agonists is greatly reduced.
Exogenously applied DHPG results in a further increase in AMPA-receptor internalisation in cultured
hippocampal neurons treated with Fmr1 specific siRNA, but this increase is far less robust than that
seen in hippocampal neurons treated with control siRNA. Taken together, the internalisation of
AMPA receptors is specifically dependent upon translation, marking it as one of the local protein
synthesis dependent consequences of group 1 mGluR-signalling and thus as one of the effects of
mGluR stimulation that should be upregulated in the absence of FMRP according to the ‘mGluR
theory of fragile X’. And indeed, after loss of FMRP the internalisation of AMPA-receptors, like local
protein synthesis, is enhanced during basal conditions and displays decreased sensitivity to
glutamate, lending support to the ‘mGluR theory of fragile X’.
Moreover, it has been suggested that the increased AMPA receptor internalisation with parallel
decrease in the number of AMPA receptors at the surface of the synapse, might also explain the
spine phenotype seen in both Fmr1 knockout mice and FXS patients (Bear et al., 2004; de Vrij et al.,
2008). Using two-photon uncaging of glutamate coupled to electrophysiology techniques to
determine the number and location of functional glutamate receptors, a tight correlation between
spine morphology and the number of functional AMPA receptors has been demonstrated in
11
pyramidal neurons of the hippocampus (Matsuzaki et al., 2001). Thus the decreased amount of
AMPA receptors at the postsynaptic density would offer an explanation for the immature spine
morphology seen in Fmr1 knockout mice and FXS patients. Since increased AMPA receptor
internalisation has been shown to be a consequence of mGluR-signalling, the ‘mGluR theory of fragile
X’ might be said to offer an explanation for the spine phenotype seen in Fmr1 knockout mice and FXS
patients through the increased AMPA receptor internalisation observed after loss of FMRP. A more
direct line of evidence for the involvement of enhanced mGluR-signalling in the spine phenotype
seen in Fmr1 knockout mice and FXS patients is the finding that treatment of cultured hippocampal
neurons with DHPG increases the proportion of thin tortuous spines (Vanderklish and Edelman,
2002). This effect of DHPG was again found to require local translation but not transcription. Thus
excessive group 1 mGluR signalling, possibly mediated by the enhanced internalisation of AMPA
receptors, could account for the spine abnormalities seen in both Fmr1 knockout mice and FXS
patients strengthening the ‘mGluR theory of FXS’.
Although brief stimulation of group 1 mGluRs facilitates the induction of LTP, more robust
stimulation induces long term depression (LTD) as well as reduces the potentiation of synaptic
transmission caused by previously established long term potentiation (LTP) (Bashir and Collingridge,
1994; Cohen and Abraham, 1996; Oliet et al., 1997; Palmer et al., 1997; Zho et al., 2002). Hence,
another area both enhanced group 1 mGluR signalling as well as abnormal AMPA receptor trafficking
would be expected to impact is synaptic plasticity. Indeed, long term depression induced by group 1
metabotropic glutamate receptor activation (mGluR-LTD) is significantly enhanced in the
hippocampus of Fmr1 knockout mice. In hippocampal slices of Fmr1 knockout mice at Schaffer
collateral synapses in the CA1 area mGluR-LTD, induced by DHPG or through electrical stimulation, is
significantly increased compared to WT littermates, while LTD dependent on NMDA receptor
activation is unchanged (Hou et al., 2006; Huber et al., 2002). Likewise, LTD at parallel fibre to
Purkinje cell synapses in the cerebellum, a form of synaptic plasticity driven by mGluR activation, is
significantly enhanced in FRM1 knockout mice compared to WT mice, while other parameters of
synaptic transmission are unaffected (Aiba et al., 1994; Koekkoek et al., 2005; Shigemoto et al.,
1994). In WT mice stable hippocampal mGluR-LTD requires local protein translation in dendrites but
not transcription, which may be how FMRP influences the mGluR-LTD (Huber et al., 2000; Snyder et
al., 2001). Similarly, stable cerebellar mGluR-LTD in WT mice requires local protein translation
(Karachot et al., 2001). On the other hand, overexpression of the human FMRP protein in either Fmr1
knockout or WT mice entirely blocks the induction of mGluR-LTD in the hippocampus (Hou et al.,
2006).
At the other end of the synaptic plasticity spectrum, long term potentiation (LTP) is significantly
reduced in several brain regions in the Fmr1 knockout mouse. In the sensorimotor, piriform and
cingulate cerebral cortex of the Fmr1 mouse thetaburst induced LTP is significantly reduced
compared to WT mice, while leaving basal synaptic transmission unchanged (Larson et al., 2005; Li et
al., 2002; Zhao et al., 2005)(Larson et al., 2005; Larson et al., 2008; Li et al., 2002; Zhao et al., 2005).
Likewise LTP in the lateral amygdala is significantly reduced in Fmr1 knockout mice, while again basal
synaptic transmission is unaffected (Suvrathan et al., 2010; Zhao et al., 2005). However the effect is
region-specific, in the CA1 area of the hippocampus LTP induced by theta-burst stimulation is equally
strong in Fmr1 knockout and WT mice (Godfraind et al., 1996; Larson et al., 2005; Li et al., 2002;
Paradee et al., 1999). The Fmr1 knockout mice do show an increased threshold for LTP in the CA1
area of the hippocampus, if a shorter theta burst train is used corresponding to threshold levels of
stimulation LTP in the Fmr1 knockout mouse is significantly reduced compared to WT mice, while
once again leaving basal synaptic transmission unaffected (Lauterborn et al., 2007).However with
respect to LTP impairment the phenotype is a lot less marked in the hippocampus than in other brain
areas like the amygdala or several regions of the cerebral cortex. A similar weaker phenotype is seen
in the prefrontal cortex, the threshold for spike-timing dependent LTP is significantly increased in the
prefrontal cortex of Fmr1 knockout mice (Meredith et al., 2007).
12
Through the effects on synaptic plasticity in the hippocampus and cerebral cortex excessive mGluR
signalling can be envisioned to explain the cognitive impairments and mental retardation
characteristics of FXS, the effects on synaptic plasiticity in the cerebellum the motor coordination
abnormalities. A last effect of group 1 mGluR stimulation on synaptic plasticity is the long term
increase in the excitability seen in neocortical layer 5 neurons (Sourdet et al., 2003). Through this
effect excessive group 1 mGluR signalling may explain both the previously discussed enhanced
audiogenic seizure sensitivity of Fmr1 knockout mice, as well as the enhanced sensory sensitivity of
FXS patients.
Local protein synthesis, including of FMRP, has been shown to be stimulated by group 1 mGluRsignalling. Excessive activity of the group 1 mGluRs has been linked to a range of different
phenotypes and FXS symptoms, ranging from abnormalities in the expression of AMPA receptors on
the plasma membrane and the dendritic spine phenotype to anomalous synaptic plasticity which in
turn has been proposed to underly such FXS symptoms as cognitive impairment and the neurological
features of FXS. In many cases the relevant consequences of group 1 mGluR signalling has been
shown to rely upon local protein translation. All supporting the ‘mGluR theory of fragile X’. However
by far the most direct and compelling evidence for the relevance of group 1 metabotropic mGluRs to
the pathophysiology of FXS comes from the study of mice with complete knockout of the Fmr1 as
well as a 50% knockdown of Grm5, which encodes for the group 1 metabotropic glutamate receptor
mGluR5 (Dolen et al., 2007). In these animals a whole range of different phenotypes seen in Fmr1
knockout mice, including phenotypes replicating neurological, physical and cellular symptoms of FXS,
are rescued. The enhanced sensitivity to audiogenic seizures seen in Fmr1 knockout mice is
significantly reduced in mice with a 50% knockdown of Grm5 next to complete Fmr1 knockout. Of
the physical features characteristic of Fmr1 knockout mice and FXS patients, the accelerated
prepubescent growth is significantly normalised in the double mutants. The spine phenotype of
increased density of long thin spines at visual cortex layer 3 pyramidal neurons is completely rescued,
with spine density becoming indistinguishable from WT mice in the double mutants. The irregular
ocular dominance plasticity seen in Fmr1 knockout mice is also completely rescued in the double
mutants. Where Fmr1 knockout mice show normal depression of the deprived eye but significantly
increased potentiation of the nondeprived eye and mice with 50% knockdown of mGluR5 expression
show decreased depression of the deprived eye but normal potentiation of the nondeprived eye, the
plasticity is completely normalised to WT levels in the double mutants. Finally the basal rate of
protein synthesis in the hippocampus, unchanged in mice with 50% Grm5 knockdown but
significantly increased in Fmr1 knockout mice, is rescued in mice with Fmr1 knockout and 50% Grm5
knockdown. Unequivocally linking activity of the group 1 mGluR mGluR5 to all these Fmr1 knockout
phenotypes.
13
4 Alleviation of FXS (phenotypes) using group 1 metabotropic glutamate receptor antagonists
According to the ‘mGluR theory of fragile X’ the absence of FMRP causes excessive signalling of group
1 mGluRs which underlies the diverse range of symptoms of fragile X. An important consequence of
this theory is that inhibition of group one mGluR activity should alleviate the symptoms and
phenotypes of FXS. This should hold true despite the fact that animals lacking functional mGluR5 also
display cognitive deficits and altered synaptic plasticity, and treatment of healthy rats with mGluR
five antagonists is associated some cognitive deficits, since in the context of FXS inhibition of the
excessive mGluR signalling should still be beneficial (Jia et al., 1998; Lu et al., 1997; Steckler et al.,
2005). If inhibition of group 1 mGluR signalling is indeed found to alleviate the symptoms and
phenotypes of FXS, this would offer not only strong support for the mGluR theory of fragile X, but of
course also have important therapeutic consequences.
Group one mGluR signalling can be inhibited in two ways, through antagonist that act extracellularly
on the receptor, or through components that inhibits the intracellular signalling cascade activated by
group one mGluRs. However, group one mGluR signalling involves many intracellular signalling
components also involved in the intracellular signalling cascades activated by many other receptors,
so inhibiting these components would most likely effect the signalling mediated by a range of
different receptors. Thus, this strategy is not very specific for the group one mGluRs, providing little
support for the ‘mGluR theory of fragile X’ if it works, and greatly increasing the likelihood of (severe)
side effects and possibly even toxicity. Hence, here I will focus on extracellular antagonist specific for
the group one mGluRs. Furthermore, due to a fear of severe motor side effects, the work on group
one mGluR antagonists for the treatment of FXS (phenotypes) has so far mainly focused on mGluR5
specific antagonists. The mGluR1 is predominantly expressed in the cerebellum, a brain region
associated with proper motor functioning (Shigemoto et al., 1992; Shigemoto and Mizuno, 2000). In
addition mGluR1 knockout mice display severe motor problems (Aiba et al., 1994; Conquet et al.,
1994). Finally, treating WT animals with antagonists specific for this receptor subtype impairs motor
function (El-Kouhen et al., 2006; Hodgson et al., 2011; Kolasiewicz et al., 2009). Conversely, mGluR5
is only very weakly expressed in the cerebellum (Shigemoto and Mizuno, 2000). In addition mGluR5
knockout mice have normal motor functioning and mGluR5 antagonist do not affect the motor
behaviour of WT animals (Lu et al., 1997; Spooren et al., 2000; Tatarczynska et al., 2001; Varty et al.,
2005; Walker et al., 2001).
In animal models the most commonly used mGluR5 specific antagonist is 2-methyl-6phenylethynylpyridine hydrochloride, referred to as MPEP. First identified as an mGluR5 specific
antagonist in an automated high-throughput screening system using through fluorescence to
measure changes in intracellular calcium levels in cell lines expressing the human metabotropic
glutamate receptor subtype 5a, MPEP was quickly shown to be a highly potent and specific inhibitor
of mGluR5 in rat primary cultured cortical neurons as well as in rat brain slices (Varney et al., 1999).
Further characterisation of MPEP demonstrate that it is also a highly potent and specific inhibitor of
mGluR5 in vivo and that it can effectively cross the blood brain barrier (Gasparini et al., 1999). MPEP
is an allosteric non-competitive inhibitor. Structurally MPEP is very different from glutamate (see
figure 1) and the binding of MPEP to mGluR5 does not affect the binding of glutamate to its ligand
binding site. In fact, resolution of the crystal structure demonstrated that the agonist binding domain
of mGluRs lies within the large N-terminal extracellular domain of these receptors, while MPEP binds
to a region within the 7-transmembrane domain of the receptor (Kunishima et al., 2000; Pagano et
al., 2000). MPEP is also an inverse agonist (Pagano et al., 2000). The group one mGluRs, like many
other receptors, show some activity in the absence of an agonist (Joly et al., 1995; Prezeau et al.,
1996). This is termed constitutive activity, and antagonists like MPEP that inhibit such agonistindependent activity as well as the agonist-dependent activity of a receptor, are called inverse
agonist. It is important to note that, although MPEP is a highly potent and selective inhibitor of
mGluR5, at higher concentrations (from a 1000-fold its IC50 and above) MPEP has been demonstrated
to lose some of its selectivity and also shows some inhibitory activity on the NMDA receptor (Lea et
al., 2005; O'Leary et al., 2000). In electrophysiological recordings of primary rat hippocampal
14
neurons, at concentrations of 20µM and 200µM (a 1000 and 10,000-fold above its IC50) MPEP
inhibited the activity of the NMDA receptors by 22.6% and 64.9% respectively.
Figure 1: The structures of glutamate and MPEP
Schematic representation of the structure of L-glutamate and 2-methyl-6-phenylethynylpyridine hydrochloride
(MPEP). Adapted from (Carroll et al., 2001).
As discussed in chapter 2, analogous to the human neurological FXS symptoms, the neurological
phenotype of the FMR1 knockout mouse includes prolonged epileptiform discharges in the
hippocampus and enhanced sensitivity to audiogenic seizures (Chen and Toth, 2001; Chuang et al.,
2005; Dolen et al., 2007; Yan et al., 2005). Both of these phenotypes are rescued by treatment with
MPEP. In hippocampal slices MPEP suppresses the bicculine-induced prolonged epileptiform
discharges seen in the knockout animals (Chuang et al., 2005). In an audiogenic seizure paradigm that
also induces audiogenic seizures in wild-type animals of the same genetic background, a single MPEP
intraperitoneal injection significantly decreases the seizure sensitivity of both FMR1 knockout and
wild-type mice compared to vehicle injected mice {{475 Yan, Q.J. 2005}}. The enhanced audiogenic
seizure sensitivity of FMR1 knockout mice is reflected in the significantly higher estimated ED 50 (the
amount of MPEP required to reduce the seizure activity by 50%) For FMR1 knockout compared to
wild-type mice. Furthermore, the increased seizure sensitivity of certain inbred strains compared to
F1 hybrids is reflected in the increased MPEP dose required to achieve seizure suppression in the
inbred strains. In another audiogenic seizure paradigm where about 60% of FMR1 knockout mice
injected with vehicle display audiogenic seizures but vehicle injected wild-type mice of the same
genetic background do not show any audiogenic seizure activity at all, a single intraperitoneal MPEP
injection of 10mg/kg or higher prevents all audiogenic seizure activity in FMR1 knockout mice in this
paradigm (Thomas et al., 2011).
This shows the mGluR5 antagonist MPEP can be successfully used to treat the enhanced audiogenic
seizure sensitivity seen in adult FMR1 knockout mice, which is in line with the ‘mGluR theory of
fragile X’. However, this does not provide especially strong support for this theory, since MPEP also
acts as an anticonvulsant in animals with normal FMRP expression (Chapman et al., 2000; Yan et al.,
2005). Thus, mGluR5 signalling may be involved in seizure activity in general, and the rescue by MPEP
of the enhanced audiogenic seizure sensitivity in FMR1 knockouts, may be because mGluR five
antagonists are beneficial to the suppression of seizures in general, rather than being indicative of
excessive mGluR signalling due to the absence of FMRP underlying the enhanced audiogenic seizure
sensitivity of FMR1 knockouts. Furthermore the effects of MPEP could also be due to aspecific effects
of MPEP, for example on the NMDA-receptor. The finding that enhanced audiogenic seizure
sensitivity is also rescued in FMR one knockout mice with a 50% reduced expression of mGluR5, more
directly demonstrates involvement of mGluR five signalling in the audiogenic seizures of FMR1
knockout mice, but can still be explained by involvement of mGluR5 signalling in seizures in general
and hence still does not provide especially strong support for the ‘mGluR theory of fragile X’,
although there is of course still therapeutic relevance (Dolen et al., 2007).
15
The other important phenotype related to enhanced sensitivity to sensory stimulation seen in FMR1
knockout mice, the deficit in the pre-pulse inhibition of the acoustic startle response, is also
completely rescued MPEP (de Vrij et al., 2008). A single intraperitoneal injection of MPEP increased
the PPI of the acoustic startle response, as measured by eye-blinking, of FMR1 knockout mice to
about wild-type levels, although it should be noted that an intraperitoneal MPEP injection increased
the PPI of wild-type mice to a similar extent. Another study found no effect of MPEP on the PPI of the
acoustic startle response, but this study used the much less sensitive whole body startle reflex as a
measure of the startle response (Thomas et al., 2011).
Another specific mGluR five antagonist, AFQ056, also completely rescues the PPI-deficit seen in
FMR1 knockout mice (Levenga et al., 2011). A single intraperitoneal injection of AFQ056 also
increases the PPI of the acoustic startle response of FMR1 knockout mice to about wild-type levels,
but unlike MPEP AFQ056 does not significantly influence the PPI of wild-type mice, providing more
definite support for the involvement of excessive mGluR5 signalling in an PPI-deficit specifically as
caused by a lack of FMRP rather than in PPI in general and thus providing true support for the mGluRtheory of FXS.
On a more structural level, the increased AMPA receptor internalisation and associated decreased
surface expression seen at the postsynaptic membrane of cultured hippocampal neurons treated
with FMR1 specific at siRNA, is rescued by acute MPEP treatment (Nakamoto et al., 2007). This is not
due to unspecific effects of MPEP on signalling by the NMDA receptor, since treatment of the
cultured hippocampal neurons with an NMDA receptor specific antagonist had no effect on the
increased AMPA receptor internalisation or the decreased surface expression caused by the
knockdown of FMRP.
Perhaps related to the rescue of the increased AMPA receptor internalisation (as discussed in
chapter 3), the dendritic spine phenotype seen in FMR1 knockout mice is also rescued by MPEP. The
significant increase in the ratio of long thin immature spines over mature mushroom spines in
cultured primary hippocampal neurons of FMR1 knockout mice is restored to wild-type levels by
MPEP treatment, but MPEP has no effect on the spine ratio of WT neurons (de Vrij et al., 2008). The
rescue by MPEP is unlikely to be due to any unspecific effects of MPEP on the NMDA receptor since
in the same study treatment with another, more specific, mGluR5 antagonist, Fenobam, also restores
the spine ratio to WT levels, while leaving the spine ratio of WT neurons unaffected. Fenobam has a
different structure from MPEP and is more specific for mGluR5, but binds to the same binding site in
the 7-transmembrane region of the mGluR5 receptor (Porter et al., 2005). Furthermore, in the same
study treatment with an NMDA receptor specific antagonist had no effect on the increased ratio of
long thin immature spines over mature mushroom spines seen in cultured primary hippocampal
neurons of FMR1 knockout mice (de Vrij et al., 2008). Indicating excessive mGluR5 signalling
underlies the dendritic spine phenotype seen in cultured hippocampal neurons of the Fmr1-knockout
mice. In another study investigating the dendritic spines of primary cultured hippocampal neurons of
FMR1 knockout mice with a different genetic background, the spine phenotype proved to be less
robust, however, the dendritic spines of hippocampal neurons of FMR1 knockout mice were
significantly longer than those of wild-type mice with the same genetic background (Levenga et al.,
2011). Treatment with yet another mGluR5 specific antagonist, AFQ056, significantly shortened the
dendritic spines of cultured hippocampal neurons of FMR1 knockout mice in a concentration
dependent manner. More counterintuitively, AFQ056 also significantly reduced the width and
density of the dendritic spines in the cultured hippocampal neurons of Fmr1-knockout mice.
However, this effect was marginal compared to the large decrease in the length of the dendritic
spines, so overall the AFQ056 successfully rescues the spine phenotype seen in cultured hippocampal
neurons of FMR1 knockout mice. Another study investigated the effects of MPEP on the spine
morphology of layer V pyramidal neurons in the primary somatosensory cortex of FMR1 knockout
mice in vivo (Su et al., 2011). In vivo acute MPEP-treatment had no effect on the dendritic spine
morphology of the pyramidal neurons. In neonatal Fmr1 knockout mice (P0) 1 and 2 week systematic
treatment with MPEP significantly rescues the increased spine length, and with 2 weeks treatment
16
MPEP restored the dendritic spine length to WT levels. In adult (6 week old) Fmr1 knockout mice
spine length of the pyramidal neurons is not significantly influenced by either 1 or 2 week systemic
MPEP treatment. The increased spine density seen in pyramidal neurons of Fmr1 knockout mice
compared to WT mice is significantly reduced by 1 and 2 week daily intraperitoneal MPEP injections
in neonatal Fmr1 knockout mice, a 2-week treatment is required to significantly reduce the spine
density of pyramidal cells of adult Fmr1 knockout mice. Lastly the significantly increased ratio of
immature thin spines over mature mushroom spines in Fmr1 knockout mice is fully rescued by daily
intraperitoneal MPEP injections for either 1 or 2 weeks in neonatal Fmr1 knockout mice, but MPEP
treatment could not rescue this phenotype in adult mice.
Taken together treatment with mGluR5-specific antagonists rescues many of the dendritic spine
morphology abnormalities seen in hippocampal neurons or in pyramidal neurons of the primary
somatosensory cortex of Fmr1 knockout mice. Indicating excessive mGluR5 signalling also underlies
this hallmark FXS phenotype and lending strong support to the ‘mGluR theory of Fragile X’. However,
the in vivo data indicates there may be a critical phase during development during which group 1
mGluR antagonist treatment is most successful, and although treatment of adult mice is still
beneficial, the rescue of the spine phenotype in adult mice is incomplete.
For the behavioural phenotypes of FMR1 knockout mice, the effects of MPEP are more controversial.
MPEP has been shown to rescue the open field phenotype (Yan et al., 2005). In FMR1 knockout mice
of either one or three months of age, a single intraperitoneal injection of MPEP rescued the open
field phenotype compared to knockout mice injected with vehicle to such a degree, that the
behaviour of the FMR one knockout mice treated with MPEP was no longer significantly different
from the behaviour of the wild-type mice in the open field. MPEP did not significantly influence the
behaviour of wild-type mice in the open field. In this same study a single intraperitoneal injection of
MPEP was also found to significantly reduce the locomotor activity of FMR1 knockout mice of one in
three months, this effect was also seen in WT mice of three months but to a much smaller degree.
However, another study found that a single intraperitoneal injection of MPEP significantly increased
the locomotor activity of WT from a dose of 40mg/kg and, of FMR1 mice from the much lower dose
of 10mg/kg onwards, leaving the effect of MPEP on locomotor activity in FMR1 knockout mice
controversial (Thomas et al., 2011).
The same study also assessed motor functioning using a rotarod and found a small but significant
impairment in FMR1 knockout mice, although this is again controversial since others found no
significant difference in the performance of FMR1 knockout and WT mice on this test (Peier et al.,
2000; Spencer et al., 2011). MPEP did not influence the performance of the knockout mice on the
rotarod, but it significantly increased the capacity of the FMR1 knockout mice to learn this motor skill
as calculated by dividing the time spent on the rotarod on the first day of testing from the time spent
on the rotarod on the last day of testing. The learning of WT mice was not affected by MPEP
treatment.
Taken together, it seems MPEP may be able to rescue the behavioural deficit seen in FMR1 knockout
mice in the open field as well as improve the motor learning of Fmr1 knockout mice in some
contexts, however neither of these findings are entirely free from controversy and the effect of
group 1 mGluR antagonists on the abnormal behaviour of Fmr1 requires further research.
As far as the Drosophila animal model of fragile X is concerned, the inhibition of group 1 mGluRsignalling has been shown to alleviate a number of important phenotypes. Treatment with MPEP
during development (i.e. during the larval stage), at concentrations several-fold below those at which
MPEP has been shown to influence NMDA-signalling, or with another mGluR antagonist with a
different mechanism of action, significantly decreased the percentage of dFMR1 flies displaying
mushroom body deficits, while leaving the mushroom bodies of control flies unaffected (McBride et
al., 2005). However, treatment with MPEP starting during adulthood was unable to rescue the
mushroom body deficit of dFMR1 knockout flies, indicating for the rescue of this particular deficit
17
there may be a critical period during development for successful treatment by inhibition of mGluRsignalling.
MPEP treatment of developing or of adult dFMR1 knockout flies significantly increased the naive
courtship levels of the knockout flies, with treatment during the larval stage being most beneficial
(McBride et al., 2005). On flies with normal levels of dFMR MPEP treatment started during
adulthood, on the other hand, significantly decreases the naive courtship levels, providing another
example of a qualitatively different response of MPEP in Fragile X models from WT animals. These
same results were seen with 3 other mGluR-signalling antagonists, making it highly unlikely that the
rescue of the courtship deficit in the dFMR1 knockout flies is due to anything other than the
inhibition of mGluR-signalling.
The cognitive deficits of dFMR1 knockout flies during immediate recall or short-term memory tests in
the conditioned courtship paradigm are rescued by MPEP treatment given either during developing
or adult stages, as well as by another mGluR antagonists given during either of these stages (McBride
et al., 2005). Apart from these cognitive deficits seen in the conditioned courtship paradigm, the
olfactory memory deficits seen in dFMR1 mutant flies are also significantly improved by MPEP
treatment during adulthood (Bolduc et al., 2008). The deficits in learning and memory seen
specifically in aged dFMR1 knockout flies are also effectively rescued by treatment with MPEP or
other mGluR-antagonists during ageing (Choi et al., 2010). Together showing that even if treatment is
only provided during adulthood, MPEP can successfully rescue the cognitive deficits observed in
Drosophila models of Fragile X.
Thus, in the Fmr1 knockout mouse model of FXS treatment with mGluR5 antagonists has been shown
to rescue both neurological and spine phenotypes. Some studies have also reported some benefits
for behavioural phenotypes, but these findings are not free of controversy. In the Drosophila model
however, behavioural phenotypes, including the cognitive problems, are also uncontroversially
rescued by MPEP-treatment. The success of mGluR5 antagonists in alleviating FXS phenotypes
provides further compelling support for the ‘mGluR theory of fragile X’ and may of course have
exiting clinical benefits, especially since in some of these examples the mGluR5 antagonists
treatment is beneficial even if treatment is started in adulthood.
18
Chapter 5: The potential role of Cyfip1 in the pathophysiology of Fragile X syndrome
Cyfip1 is a known cytoplasmic interactor of FMRP (Schenck et al., 2001). It is highly conserved
amongst species, with orthologs in mouse, drosophila and C. Elegans (Chai et al., 2003; Jiang et al.,
2008). It has a complicated gene structure and many different splice variants are known. It is highly
expressed throughout the CNS, in adulthood as well as during development (Bittel et al., 2006; Jiang
et al., 2008; van der Zwaag et al., 2010). The possible role of Cyfip1 in the pathophysiology of FXS has
not received much attention. However, there is some evidence from genetic and clinical studies for a
role of Cyfip1 in several of the symptoms seen in FXS. Furthermore recent data on the role of Cyfip1
in the regulation of local mRNA translation in complex with FXS points to a key role for Cyfip1 within
the ‘mGluR theory of fragile X’, mechanistically linking Cyfip1 to FXS.
Cyfip1 has been linked to neurodevelopmental syndromes with considerable overlap in their
symptoms with the Fragile X Syndrome. The Prader-Willi Syndrome (PWS) is a neurodevelopmental
disorder that like FXS is characterised by mental retardation and intellectual disabilities. Other
hallmark features include severe hypotonia, hyperphagia, which can become life-threatening if food
intake is not strictly controlled, feeding difficulties early in life, behavioural problems, autistic
behaviours and compulsive behaviours like tantrums or hoarding (Butler and Thompson, 2000; Holm
et al., 1993; Whittington and Holland, 2004).
It is well established that PWS is caused by decreased expression of paternally expressed genes from
the 15q11-q13 region of human chromosome 15, although which of these genes play a significant
role and whether the most important ones are protein coding or are non-coding small nucleolar
RNAs is not yet known (Jiang et al., 2008). The 15q11-q13 region is one of the most polymorphic
regions of the human genome, with huge variability in copy number as well as in gene organisation.
The region displays very high genomic instability, mainly because it contains a series of low copy
repeat sequences or segmental duplication sites which are recombination hotspots.
In fact, these repeat sequences or duplication sites mark the breakpoints where in about 70% of PWS
patients a paternal deletion is found (Bittel and Butler, 2005; Doornbos et al., 2009; Nicholls and
Knepper, 2001). One breakpoint (BP3) is located in the telomeric region of the proximal long arm of
the chromosome, while another two (BP1 and BP2) are located in the centromeric region. On the
basis of the size of the parental deletion two subgroups can be distinguished, the larger deletion
occurs between BP1 and BP3 and is termed a type I deletion, whereas the smaller type II deletion
takes place between BP2 and BP3. Thus, only PWS patients with a type I deletion are
haploinsufficient for the 4 genes that lie between BP1 and BP2; next to CYFIP1, GCP5, NIPA1 and
NIPA2 (Chai et al., 2003). The 4 genes are not imprinted, highly conserved across species, all have
orthologs on mouse chromosome 7C, and CYFIP1 and NIPA1/2 are widely expressed in the CNS while
expression of GCP5 is more restricted to the subthalamic nuclei. Significantly decreased mRNA levels
for the genes between BP1 and BP2 in PWS patients with a type I deletion compared to those with a
type II deletion have been confirmed (Bittel et al., 2006).
An initial study comparing the two deletion subtypes suggested PWS patients with a type I deletion
have a more severe phenotype than those with a type II deletion, implicating CYFIP1 and the other 3
genes additionally deleted in the type I deletion in PWS symptoms (Butler et al., 2004). This study,
performed among 12 patients with type I deletions and 14 with type II and using several validated
psychological and behavioural tests conducted by trained investigators blind to genetic status, found
type I patients had significantly more and/or more severe behavioural and psychological problems,
including lower adaptive behaviour scores and more severe compulsions, as well as significantly
decreased reading, maths and visual motor integration abilities. Another study, slightly larger but still
with only 14 type I deletion patients, was not able to replicate all of these findings, although this
study did find the verbal IQ to be significantly decreased in type I compared to type II deletion
patients and found a non-significant trend towards a poorer performance by type I compared to type
II deletion patients in all tests of ability that may become significant with larger sample sizes,
implying type I deletion patients have more severe intellectual problems as was also seen in the
19
study by Butler et al (Milner et al., 2005). Two other studies, including one larger study with 26 type I
and 29 type II deletion patients, were unable to detect any significant differences in the behavioural,
psychological or intellectual abilities of type I compared to type II deletion patients (Dykens and Roof,
2008; Varela et al., 2005). However these studies, unlike the previous two, did not use trained
investigators blind to the genetic status to assess the behavioural, psychological or psychological
problems of the patients, but instead relied entirely on the information provided by the parents or
primary caregivers, perhaps making the findings of these studies less reliable. Taken together there
are some indications Cyfip1 and the other 3 genes lying between breakpoint 1 and 2 in the 15q11q13 region may be involved in the worsening of PWS symptoms in type I deletion patients, but this
remains a controversial issue.
Linking the four genes between breakpoints 1 and 2 more convincingly to PWS symptoms is the
finding that messenger RNA levels correlate positively with a range of behavioural, psychological and
intellectual assessment values (Bittel et al., 2006). The amount of mRNA for the four genes expressed
was found to explain between 24 and 99% of the variation in the behavioural, psychological and
academic measures examined, as indicated by the coefficient of determination, while deletion type (I
or II) was found to explain between 5 and 50%. The quantity of Cyfip1 mRNA was found to explain up
to 60% of the assessed parameters, thus linking CYFIP1 much more robustly to behavioural,
psychological and academic problems seen in PWS patients.
Apart from some overlap in symptoms there is another link between Fragile X and Prader-Willi
syndrome. A subset of FXS patients have been described that on top of the FXS characteristics also
display many of the PWS symptoms that are not normally seen in FXS patients, FXS patients with the
so-called Prader-Willi-like Phenotype (PWP) (de Vries et al., 1993; Nowicki et al., 2007; SchranderStumpel et al., 1994)(). Genetically the patients display the Fmr1 abnormalities characteristic of FXS
patients but lack the 15q11-q13 region abnormalities seen in PWS patients, underscoring the validity
of classing PWP as a subtype of FXS with PWP-like symptoms rather than as a subtype of PWS with
FXS-like symptoms. The described PWP patients all develop the hallmark PWS symptoms of severe
hyperphagia during childhood, leading to extreme obesity if food intake is not strictly controlled.
They also all show a remarkable number of other PWS symptoms not seen in FXS patients without
PWP, but the specific symptoms differ amongst patients. In a study examining 13 patients with PWP,
the expression of CYFIP1 mRNA was found to be dramatically and significantly decreased in these
patients compared to patients with FXS without PWP or in healthy controls, suggesting CYFIP1 is
important in the pathophysiology of PWP (Nowicki et al., 2007).
Many FXS patients also display autistic behaviour and it is also seen in PWS patients. Among FXS
patients with PWP autism is even more common, which may be linked to their abnormal gene
expression of CYFIP1 (Nowicki et al., 2007). A microduplication involving CYFIP1 and the three other
genes between BP1 and BP2 of chromosome 15 was found to co-segregate with autism in a family
(van der Zwaag et al., 2010). The duplication resulted in a significant increase in the mRNA expression
of these genes and although the duplication was also seen in some unaffected controls and the
control group was too small for the increased occurrence of the microduplication in patients to reach
statistical significance, this still points to CYFIP1 as a potential risk gene for autism. Another study
found CYFIP1 mRNA expression is selectively and significantly upregulated in FXS patients diagnosed
with autism as well as in patients with autism caused by maternal 15q11-q13 duplication compared
to non-autistic controls (Nishimura et al., 2007). Thus autism, including the autistic behaviour seen in
some FXS patients, is another neurodevelopmental disorder CYFIP1 has been linked to.
Interestingly, another set of patients have been described with a microdeletion spanning just the 4
genes between BP1 and BP2. Lacking abnormalities between BP2 and BP3 these patients do not have
PWS but they do display a range of problems, many analogous to PWS or FXS symptoms. Initially a
case study of a single boy with the microdeletion was published (Murthy et al., 2007). The boy, 3 ½
years old at the time, displayed severe mental retardation, neurological problems, developmental
delay and severe speech impairment. He was found to have inherited the deletion from his father,
who had the same microdeletion but was much less severely affected than his son, highlighting the
incomplete penetrance of the disorder. These findings were replicated in a larger study describing 9
20
patients with the microdeletion, all showing developmental delay, especially delayed motor and
speech development, dysmorphisms and behavioural problems such as ADHD, autism or obsessive
compulsive symptoms, although the specific symptoms and severity of the disorder differed amongst
patients and there was incomplete penetrance as the microdeletion was often inherited from a
mildly affected, or even unaffected, parent (Doornbos et al., 2009). The microdeletion was not found
in 350 healthy non-related controls and although this did not constitute a large enough control group
to reach statistical significance the authors conclude the microdeletion has a pathogenic nature and
at least contributes to the observed phenotype. Once again linking CYFIP1 and the other three genes
located between BP1 and BP2, to problems such as developmental delay and behavioural issues like
autism and obsessive-compulsive behaviours also seen in FXS.
Local translation in the dendrites of neurons is known to be especially reliant upon cap-dependent
translation (Richter and Sonenberg, 2005). Cap-dependent translation requires the assembly of
eukaryotic initiation factor 4A (eIF4A), eIF4G and eIF4E into a complex known as eIF4F on the 5’
terminal cap of mRNAs. Moreover poly-A-binding protein (PABP), which binds to the poly-A-tail
present at the 3’ terminal end of mRNA’s, interacts with the protein complex present on the 5’ cap,
circularising the mRNAs (Mazumder et al., 2003). Through a series of immunoprecipitation (IP),
Sepharose chromatography and pull down experiments Napoli et al have shown a protein complex
containing CYFIP1, FMRP, eIF4E and PABP is present on neuronal mRNAs (Napoli et al., 2008). In
m7GTP chromatography, using Sepharose beads coupled to the mRNA cap analogue m 7GTP, only
specific elution recovered CYFIP1, FMRP, eIF4E and PABP from the beads after incubation with
mouse cytoplasmic total brain extract, while 2 proteins not involved in protein translation remained
bound to the beads after specific elution as did the cytoplasmic CYFIP1 interactor WAVE. In pull down
experiments using total brain extract it was possible to pull down PABP, CYFIP1, FMRP and to a lesser
degree eIF4E with poly(A)-Sepharose beads. In IP experiments using total brain extract CYFIP1 and
eIF4E were shown to be co-precipitated by FMRP immunoprecipitation, while in CYFIP1 IP
experiments FMRP, eIF4E and PABP were shown to co-precipitate. Furthermore using cytoplasmic
total brain extract from FMR1-knockout mice they were able to show the eIF4E-CYFIP1-PABP protein
complex can assemble on the cap of mRNAs in the absence of FMRPs, since CYFIP1 and eIF4E were
still recovered from m7GTP-Sepharose beads by specific elution only and CYFIP1 and PABP were still
captured by poly(A)-Sepharose beads after incubation with brain extract from FMR1 knockout mice.
One way the FMRP-CYFIP1-eIF4E-PABP complex assembled on the cap of mRNAs could repress
translation is by inhibiting the assembly of eIF4F. This is the way eIF4E Binding Proteins (4E-BPs)
work, by binding eIF4E they interfere with the eIF4E-eIF4G association, thereby inhibiting the
assembly of the eIF4F complex on the cap of mRNAs and so repressing cap-dependent translation of
the mRNA. Several of the characterised 4E-BPs and eIF4G share a short motif responsible for their
association with eIF4E (Richter and Sonenberg, 2005). To determine if CYIP1 might be a 4E-BP Napoli
et al performed a multisequence alignment search to see if such a motif is contained within the
CYFIP1 sequence (Napoli et al., 2008). Indeed, in the human CYFIP1 sequence a candidate binding
peptide is found at residue 733-751. Furthermore, the predicted structural arrangement of this
candidate peptide shows it could potentially adopt the ‘reverse L-shaped conformation’ adopted by
human 4E-BP1 when it is complexed with eIF4E, and in this confirmation would fit the binding pocket
of eIF4E (Napoli et al., 2008; Tomoo et al., 2005). To determine if CYFIP1 is in fact a 4E-BP, an in vitro
binding experiment was conducted with GST-eIF4E coupled to Sepharose beads and in vitro
synthesised radioactively-labelled CYFIP1 and FRMP proteins. CYFIP1 was specifically and efficiently
precipitated by GST-eIF4E while FMRP was not, indicating CYFIP1, but not FMRP, directly and
efficiently binds to eIF4E. In m7GTP-Sepharose chromatography experiments in vitro synthesized
CYFIP1 and eIF4E were present in the specific eluate only, indicating CYFIP1 still binds directly to
eIF4E when eIF4E is also bound to the cap of mRNAs. A series of mutated constructs showed the
candidate binding peptide is indeed the area of CYFIP1 responsible for its interaction with eIF4E. The
‘Reverse L-shaped structure’ has two α-helices at its centre and is stabilised by two internal salt
bridges. If CYFIP1 is mutated at the site of the two internal saltbridges, which would disrupt the
21
‘reverse L-shaped confirmation’, the association of CYFIP1 with eIF4E in m7GTP-Sepharose
chromatography experiments is significantly reduced. The predicted structure of the CYFIP1
candidate peptide also pointed to Lys743 as an amino acid ideally positioned for direct interaction
with eIF4E. If this single amino acid is mutated, which does not disrupt the ‘reverse L-shaped
confirmation’ of the candidate binding peptide, the interaction of CYFIP1 with eIF4E is dramatically
reduced in the m7GTP-Sepharose chromatography experiments. Pull down experiments using in vitro
synthesized WT and mutated versions of the candidate binding peptide confirmed Lys743 is critical
for the interaction between CYFIP1 and eIF4E, while the internal saltbridges function to stabilise the
‘reverse L-shaped confirmation’ maintaining Lys743 in a confirmation favourable for its interaction
with eIF4E. In conclusion Cyfip1 is a 4E-BP that interacts with eIF4E through a domain with structural
homology to the other 4E-BPs.
In Cyfip1 IP experiment using cytoplasmic extract from primary cortical neurons FMRP and eIF4E
readily co-precipitate with the Cyfip1, but eIF4G does not, indicating Cyfip1 like other 4E-BPs
competes with eIF4G for the binding of eIF4E. Thus, CYFIP1 would be expected to repress the
translation of mRNAs. This was tested by influencing the expression of CYFIP1 in human HeLa cells.
Overexpression of CYFIP1 causes a significant decrease in the protein production of cells as measured
by quantifying the total amount of radioactively labelled protein produced by cells normalised to the
amount of radioactively-labelled GAPDH, a protein that is constitutively expressed by HeLa cells,
produced by the cells. On the other hand decreasing the expression of CYFIP1 through the use of
siRNAs significantly increases the total amount of protein produced by cells. Finally, a CYFIP1
construct was targeted to a specific mRNA in the HeLa cells. Overexpression of this construct
decreased the translation of the mRNA the CYFIP1 was targeted to significantly further than
overexpression of an untargeted version of CYFIP1, which already caused a significant reduction in
the synthesis of the protein compared to transfection with a control vector. In conclusion in vivo
CYFIP1 functions as a general inhibitor of translation in mammalian cells.
However in the brain Cyfip1 is found in a protein complex on the cap of mRNAs together with FMRP,
so potentially FMRP might be involved in recruiting Cyfip1 to the cap of mRNAs which might make its
repression of translation more limited to FRMP-target mRNAs. In in vitro binding experiments using
GST-eIF4E coupled to Sepharose beads and in vitro synthesised radioactively labelled Cyfip1 and
FMRP proteins the presence of the capped FMRP-target mRNA Arc/Arg3.1 causes a significant
increase in the amount of Cyfip1 precipitated by the GST-eIF4E and whereas in the absence of
capped mRNA FMRP was not efficiently precipitated at all, when capped Arc/Arg3.1 is added FMRP is
also efficiently precipitated by the beads. In contrast the presence of capped non-FMRP-target
luciferase mRNA only causes a faint increase in the amount of Cyfip1 and FMRP precipitated by GSTeIF4E. Suggesting, the interaction between Cyfip1-FMRP and eIF4E is especially increased by the
presence of capped FMRP-target mRNAs other capped mRNAs also have some effect. Furthermore
Cyfip1 IP experiments in combination with real time or reverse transcriptase polymerase chain
reaction (RT-PCR) in mouse cytoplasmic brain extracts reveal that Cyfip1 forms ribonucleoprotein
(RNP) particles with the FMRP-target mRNAs Map1b, App, αCaMKII and again Arc/Arg3.1, while the
non-FMRP-target mRNA D2DR could not be detected in the Cyfip1 associated complex. Suggesting
Cyfip1 is specifically associated with FMRP-target mRNAs in neurons, although the FMR1 mRNA
which is also a FMRP-target mRNA could not be detected in Cyfip1 associated complex indicating
Cyfip1 may associate with many but does not associate with all FMRP-target mRNAs in neurons. The
non-coding BC1 RNA enhances the association of FMRP with some of its target mRNAs(Zalfa et al.,
2003). It is also found to be part of the Cyfip1-associated complex in the CYFIP1 IP experiments in
mouse cytoplasmic brain extracts. If the Cyfip1 IP and RT-PCR experiments are repeated using brain
extracts from BC1 knockout mice the association of Cyfip1 with the FMRP-target mRNAs Map1b,
App, αCaMKII and Arc/Arg3.1 is significantly reduced, again hinting to a role for FMRP in recruiting
Cyfip1 to the cap of mRNAs. Indeed, in CYFIP1 IP experiments in brain extracts of Fmr1 knockout
mice the association of Cyfip1 with Map1b is significantly reduced which is confirmed using
quantitative RT-PCR (RT-Q-PCR). Decreasing the expression of CYFIP1 in mammalian primary cortical
neurons through electroporation of CYFIP1 siRNAs significantly increases the translation of the
22
FMRP-target mRNA MAP1B, as determined by immunoblotting followed by quantification and
normalisation to Akt1 expression levels. In contrast, the expression of Vinculin, encoded for by a nonFMRP-target mRNA, is not significantly influenced by the decreased Cyfip1 levels. Likewise,
immunoblotting of cytoplasmic total brain extracts of CYFIP1+/- heterozygous knockout mice revealed
that after normalisation for LDH the protein levels of Map1b, APP and αCaMKII, all coded for by
FMRP-target mRNAs, were significantly increased compared to WT mice, while the protein levels of
Vinculin were again unaffected. However, both in total brain extract of CYFIP1 heterozygous
knockout mice and in primary cortical neurons transfected with CYFIP1 siRNAs the protein expression
levels of FMRP remained unaffected by the decreased Cyfip1 levels, as was to be expected since the
Fmr1 mRNA could also not be detected in the Cyfip1-associated complex by RT-PCR following Cyfip1
immunoprecipitation in brain extracts. In conclusion, in neurons Cyfip1 requires BC1 RNA and the
FMRP for optimal association with mRNA and it shows enhanced association and so also enhanced
translational repression of a subset of FMRP-target mRNAs, but not of Fmr1 mRNA which is also a
FMRP-target mRNA.
Finally Napoli et al were also able to show that in neurons the association of the Cyfip1 containing
protein complex on the cap of mRNAs and thus of the translational regulation of Cyfip1, can be
regulated by synaptic activity. In primary hippocampal neurons eIF4E colocalises with Cyfip1 and
FMRP in cell bodies and dendrites. This colocalisation is significantly reduced after the neurons were
stimulated for 30’ with brain derived neurotrophic factor (BDNF) as after a 1h stimulation, although a
4h stimulation did not significantly affect the colocalisation. Stimulation with BDNF for 30’ also
significantly decreases the amount of eIF4E coprecipitated in Cyfip1 IP experiments using primary
hippocampal neuron extracts, indicating the decreased colocalisation seen in primary hippocampal
neurons upon BDNF stimulation reflects a decrease in Cyfip1- eIF4E interactions. Using
synaptoneurosomes they showed a similar regulation takes place at synapses. In Cyfip1 IP
experiments the amount of eIF4E coprecipitated in extracts of synaptoneurosomes stimulated for 30’
with BDNF is significantly reduced compared to in extracts of unstimulated synaptoneurosomes. RTQ-PCR data indicate the amount of Map1b mRNA and BC1 RNA coprecipitating in Cyfip1 IP
experiments is also dramatically reduced in extracts of synaptoneurosomes exposed to BDNF for 30’
compared to in extracts of unexposed synaptoneurosomes. Stimulation of synaptoneurosomes with
the group 1 mGluR agonist DHPG had the same effect of decreased coprecipitation of eIF4E with
Cyfip1, but only after a brief 5’ stimulation, while a 15’ stimulation with DHPG actually significantly
increases the coprecipitation efficiency of eIF4E in synaptoneurosomes.
23
Conclusion
Fragile X Syndrome, the most common form of mental retardation, is caused by disruptions to a
single gene, Fmr1. In nearly all FXS patients the expression of the protein encoded for by Fmr1,
FMRP, is lost; most commonly due to an expansion of a CGG repeat in the 5’-untranslated region of
FMR1 leading to excessive methylation of the gene (Fu et al., 1991; Hirst et al., 1995; Meijer et al.,
1994; Oberle et al., 1991; Sutcliffe et al., 1992; Verkerk et al., 1991). There is only a single exception
where FMRP expression is not lost and yet the patients display an especially severe form of FXS (De
Boulle et al., 1993). In this case the FMRP protein contains a single point mutation resulting in one
amino acid substitution (Siomi et al., 1994). This mutation does not influence the ability of FMRP to
bind mRNA molecules in vivo, but instead specifically impairs its ability to associate with
polyribosomes (Feng et al., 1997a). Strongly suggesting that, although FMRP has a number of
different functions in cells such as in mRNA transport as a kinesin adaptor and in the regulation of
mRNA expression as a part of the RNA interference machinery, it is its role in the regulation of (local)
mRNA translation as a binding partner of polyribosomes, mainly repressing the translation of its
target mRNAs, that is crucial to the pathophysiology of FXS (Laggerbauer et al., 2001; Lu et al., 2004).
As a mechanistic explanation of the pathophysiology of FXS the ‘mGluR theory of FXS’ is most
generally accepted (Bear et al., 2004). According to this theory the activation of group 1
metabotropic glutamate receptors (mGluRs) at dendrites stimulates the local translation of mRNAs
that are responsible for the functional effects of mGluR-signalling as well as the translation of FMRP.
The FMRP inhibits the further local translation of these mRNAs, serving as a form of end-product
inhibition for the mGluR-signalling. In the absence of FMRP, as in FXS, the inhibition by FMRP falls
away leading to an exaggeration of all local protein synthesis-dependent effects of mGluR-signalling.
According to the theory this excessive level of basal group 1 mGluR-signalling, as well as a decreased
responsiveness to glutamate due to the elevated basal signalling levels, is the underlying cause of the
wide range of symptoms seen in FXS. So in line with the genetic and clinical data discussed above, the
‘mGluR theory of FXS’ also points to the role of FMRP in the regulation of local mRNA translation as
critical to the pathophysiology of FXS.
The Fmr1 knockout mouse model of FXS displays a multifaceted phenotype that is generally consist
with the symptoms seen in FXS patients and includes phenotypes analogous to the physical,
neurological, structural and behavioural characteristics of FXS in humans (Bakker et al., 1994).
However, the hallmark mental retardation does not seem to be as effectively replicated in the Fmr1
knockout mouse. Instead this key feature of the disorder appears to be more effectively replicated in
another animal model of FXS, the dFmr1 loss of function Drosophila mutant (Bolduc et al., 2008; Choi
et al., 2010; McBride et al., 2005; Wan et al., 2000).
Data gathered from both the mouse and the Drosophila model of FXS supports the ‘mGluR theory of
fragile X’. The stimulation of group 1 mGluRs results in the increased local translation of FMRP
(Ferrari et al., 2007; Todd et al., 2003a; Todd et al., 2003b; Weiler et al., 2004). The rate of basal local
mRNA translation is significantly elevated in the cortex and hippocampus of the Fmr1 knockout
mouse (Berry-Kravis et al., 2011; Dolen et al., 2007; Hou et al., 2006; Qin et al., 2005). On the other
hand the increase in local protein synthesis in response to group 1 mGluR stimulation through
agonists or by the induction of mGluR-dependent LTD is significantly reduced in Fmr1 knockout mice
(Berry-Kravis et al., 2011; Greenough et al., 2001; Hou et al., 2006; Weiler et al., 2004). In the cortex
and the amygdala of the Fmr1 knockout mouse the internalisation of AMPA-receptors is increased,
which is another local mRNA-translation dependent consequence of mGluR-signalling (Li et al., 2002;
Suvrathan et al., 2010). Through the increased AMPA-receptor internalisation the ‘mGluR theory of
fragile X’ may also explain the spine phenotype of FXS as spine geometry has been shown to be
tightly correlated to the expression of AMPA receptors at its surface, but a more direct line of
evidence comes from the fact that the treatment of cultured hippocampal neurons with a group 1
mGluR antagonists increases the proportion of thin immature spines(Bear et al., 2004; Matsuzaki et
al., 2001; Vanderklish and Edelman, 2002). Finally, Fmr1 knockout mice show synaptic plasticity
abnormalities in a range of different brain structures, mainly increased mGluR-dependent LTD and
24
decreased LTP, which can of course be linked to excessive basal mGluR-signalling levels supporting
the ‘mGluR theory of Fragile X’ (Aiba et al., 1994; Hou et al., 2006; Huber et al., 2002; Koekkoek et al.,
2005; Larson et al., 2005; Lauterborn et al., 2007; Li et al., 2002; Meredith et al., 2007; Shigemoto et
al., 1994; Suvrathan et al., 2010; Zhao et al., 2005). Synaptic plasticity abnormalities could underlie
many of the behavioural, cognitive and neurological symptoms of FXS.
But most convincingly, in mice with complete knockout of Fmr1 as well as a 50% knockdown of
Grm5, the mRNA coding for the group 1 mGluR mGluR5, a whole range of the different phenotypes
seen in Fmr1 knockout mice, including phenotypes replicating neurological, physical and cellular
symptoms of FXS, are significantly rescued (Dolen et al., 2007). Directly proving the relevance of
excessive group 1 mGluR-signalling to these FXS symptoms. Along these same lines, inhibiting group
1 mGluR-signalling through pharmacological methods significantly reduces or even completely
rescues phenotypes analogous to several neurological, cellular and behavioural characteristics of FXS
(Chuang et al., 2005; de Vrij et al., 2008; Levenga et al., 2011; Su et al., 2011; Yan et al., 2005). In the
Drosophila FXS model inhibiting group 1 mGluR signalling was also able to rescue many of the
cognitive deficits seen in these flies (Bolduc et al., 2008; Choi et al., 2010; McBride et al., 2005).
Alleviation of such a diverse range of symptoms by the inhibition of group 1 mGluR-signalling in two
different animal models of FXS establishes the validity of the main points of the ‘mGluR theory of
Fragile X’, including the assumption that the main role of FMRP in local protein translation is crucial
to the pathophysiology of FXS. Cyfip1 is a known cytoplasmic interactor of FMRP (Schenck et al.,
2001). Genetic and clinical studies have hinted at a role for Cyfip1 and the 3 other genes lying
between breakpoint 1 and breakpoint 2 on chromosome 15 in the worsening of the Prader-Willi
Syndrome (PWS), a syndrome with considerable overlap in symptoms with FXS, in patients with a
type I deletion (Butler et al., 2004; Milner et al., 2005). Although these findings remain controversial,
a microdeletion spanning just these 4 genes has also been linked to a disorder with considerable
overlap in symptoms with FXS further suggesting a role for Cyfip1 in these symptoms, although the
microdeletion was also found in some relatively mildly or unaffected individuals and this study also
did not reach statistical significance (Doornbos et al., 2009; Murthy et al., 2007). More convincing is
the finding that Cyfip1 expression is significantly reduced in patients with a rare but severe subtype
of FXS, the Prader-Willi-like Phenotype (PWP) (Nowicki et al., 2007). PWP patients have Fmr1 genetic
abnormalities characteristic of FXS, but lack the 15q11-q13 abnormalities seen in PWS patients yet
display several symptoms of PWS not usually seen in FXS patients on top of unusually severe FXS.
Thus implicating the decreased Cyfip1 expression, not only in the PWS symptoms seen in PWP
patients, but also in the aggravation of FXS symptoms. Lastly Cyfip1 has been proposed as a risk gene
for autism, although this is based on data that did not reach statistical significance, but again more
convincingly the expression of Cyfip1 has been shown to be significantly increased in FXS patients
with autism, as well as in autism patients without FXS with a specific form of autism, compared to
non-autistic controls (Nishimura et al., 2007; van der Zwaag et al., 2010).
Thus, there is some evidence from genetic studies that Cyfip1 might play a role in some of the
symptoms of FXS. However, it is mechanistically that the case for a key role of Cyfip1 in the
pathophysiology of FXS becomes really convincing. A mutated form of FXS that shows normal
expression and mRNA binding but is specifically impaired in the binding of polyribosomes leads to an
especially severe form of FXS and the ‘mGluR theory of FXS’, for which data on animal models
provides many lines of support most importantly alleviating of FXS symptoms by inhibition of group 1
mGluR-signalling through genetic or pharmacological methods, also points to the role of FMRP as
repressor of the local translation of specific mRNAs as key to the pathophysiology of FXS. Cyfip1 has
been shown to mediate this inhibition of local mRNA translation by FMRP through its function as a
4E-BP (Napoli et al., 2008). Cyfip contains a motif with structural homology to other 4E-BP and eIF4G
and through this motif it interacts with eIF4E in vivo. The binding of Cyfip1 to a mRNA-molecule
significantly reduces its translation in vivo and FMRP helps to recruit CYFIP1 to the cap of its targetmRNAs. Thus, the reduction of Cyfip1 expression, as in CYFIP1+/- heterozygous knockout mice,
significantly increases the translation of a variety of different FMRP-target mRNAs, although not for
all FMRP-target mRNAs, namely not for Fmr1-mRNA. This regulation of translation through
25
cooperation of Cyfip1 and FMRP is regulated by synaptic activity. Thus for many of the FMRP-target
mRNAs, including some key brain mRNAs that would be expected to be of key importance for many
of the local protein-synthesis dependent consequences of mGluR-theory, it is in fact Cyfip1 that is
mediating the effects of FMRP on local mRNA translation. Thus placing CYFIP1, just like FMRP, at the
heart of the ‘mGluR theory of FXS’ and warranting a much more extensive investigation into the role
of Cyfip1 in the pathophysiology of FXS.
26
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