Correction of Fragile X Syndrome in Mice

Article (PDF Available)inNeuron 56(6):955-62 · January 2008with66 Reads
DOI: 10.1016/j.neuron.2007.12.001 · Source: PubMed
Abstract
Fragile X syndrome (FXS) is the most common form of heritable mental retardation and the leading identified cause of autism. FXS is caused by transcriptional silencing of the FMR1 gene that encodes the fragile X mental retardation protein (FMRP), but the pathogenesis of the disease is unknown. According to one proposal, many psychiatric and neurological symptoms of FXS result from unchecked activation of mGluR5, a metabotropic glutamate receptor. To test this idea we generated Fmr1 mutant mice with a 50% reduction in mGluR5 expression and studied a range of phenotypes with relevance to the human disorder. Our results demonstrate that mGluR5 contributes significantly to the pathogenesis of the disease, a finding that has significant therapeutic implications for fragile X and related developmental disorders.
Neuron
Report
Correction of Fragile X Syndrome in Mice
Gu¨lDo
¨
len,
1,2
Emily Osterweil,
1
B.S. Shankaranarayana Rao,
3
Gordon B. Smith,
1
Benjamin D. Auerbach,
1
Sumantra Chattarji,
4
and Mark F. Bear
1,
*
1
Howard Hughes Medical Institute, The Picower Institute for Learning and Memory, Department of Brain and Cognitive Sciences,
Massachusetts Institute of Technology, Cambridge, MA 02139, USA
2
Department of Neuroscience, Brown Medical School and the Division of Biology and Medicine, Providence, RI 02912, USA
3
Department of Neurophysiology, National Institute of Mental Health and Neuroscience, Bangalore 560 002, India
4
National Center for Biological Sciences, Tata Institute of Fundamental Research, Bangalore 560 002, India
*Correspondence: mbear@mit.edu
DOI 10.1016/j.neuron.2007.12.001
SUMMARY
Fragile X syndrome (FXS) is the most common
form of heritable mental retardation and the
leading identified cause of autism. FXS is caused
by transcriptional silencing of the FMR1 gene
that encodes the fragile X mental retardation
protein (FMRP), but the pathogenesis of the dis-
ease is unknown. According to one proposal,
many psychiatric and neurological symptoms
of FXS result from unchecked activation of
mGluR5, a metabotropic glutamate receptor.
To test this idea we generated Fmr1 mutant
mice with a 50% reduction in mGluR5 expres-
sion and studied a range of phenotypes with rel-
evance to the human disorder. Our results dem-
onstrate that mGluR5 contributes significantly
to the pathogenesis of the disease, a finding
that has significant therapeutic implications for
fragile X and related developmental disorders.
INTRODUCTION
Despite progress understanding the etiology of fragile X, it
is still unknown how disruption of brain function by the
FMR1 mutation leads to a devastating syndrome that
includes altered neural development, cognitive impair-
ment, childhood epilepsy, and autism (Bernardet and Cru-
sio, 2006). There is no treatment for fragile X syndrome
(FXS), and the prospects for therapy by gene replacement
are not promising (Peier et al., 2000). Future therapeutic
approaches must therefore be based on a more complete
understanding of the basic pathogenesis of the disease.
FMRP is enriched postsynaptically in the brain, particu-
larly at synapses that use the major excitatory neurotrans-
mitter glutamate, so much attention has been focused on
synaptic dysfunction in FXS. Recently a ‘metabotropic
glutamate receptor (mGluR) theory’ of fragile X pathogen-
esis was proposed (Bear et al., 2004), based on the follow-
ing four observations: (1) FMRP can function as a repressor
of mRNA translation at synapses (Brown et al., 2001; Qin
et al., 2005); (2) synaptic protein synthesis is stimulated
potently by activation of group 1 (Gp1) mGluRs, compris-
ing mGluR1 and mGluR5 (Weiler and Greenough, 1993);
(3) many of the lasting consequences of activating Gp1
mGluRs depend on synaptic mRNA translation (Huber
et al., 2000; Karachot et al., 2001; Merlin et al., 1998; Ray-
mond et al., 2000; Vanderklish and Edelman, 2002; Zho
et al., 2002); and (4) in the absence of FMRP, several pro-
tein synthesis-dependent consequences of activating
mGluRs are exaggerated (Chuang et al., 2005; Hou et al.,
2006; Huber et al., 2002; Koekkoek et al., 2005). Together,
these findings have led to the idea that FMRP and Gp1
mGluRs normally work in functional opposition, and that
in the absence of FMRP, unchecked mGluR-dependent
protein synthesis leads to the pathogenesis of FXS (Fig-
ure S1 available online).
The appeal of the mGluR theory stems from its simplic-
ity and the potentially profound therapeutic implication—
that downregulating Gp1 mGluR signaling could correct
multiple symptoms of FXS. However, the theory remains
controversial. To date, the strongest evidence in favor of
the mGluR theory (McBride et al., 2005; Tucker et al.,
2006) has been indirect, relying on drug treatments in non-
mammalian species with mGluR orthologs coupled to dif-
ferent signaling cascades than mammalian Gp1 mGluRs
(Bjarnadottir et al., 2005). It has been shown in fragile X
knockout mice that acute administration of MPEP
[2-methyl-6-(phenylethynyl)-pyridine], an mGluR5 antago-
nist, can reversibly suppress seizure phenotypes (Chuang
et al., 2005; Yan et al., 2005). However, in addition to off-
target activity of MPEP (Heidbreder et al., 2003; Lea and
Faden, 2006), interpretation of this finding is complicated
by the fact that the drug is anticonvulsant in wild-type mice
as well. Thus, it remains to be established if chronic down-
regulation of Gp1 mGluR signaling can correct altered
development in fragile X, as predicted by the mGluR
theory. In the current study, we used a genetic strategy
to definitively address this critical question.
RESULTS
Rescue Strategy and Rationale
Because both the human FMR1 and GRM5 genes have
functional homologs in the mouse (Fmr1 and Grm5), we
were able to generate Fmr1 knockout mice with reduced
Neuron 56, 955–962, December 20, 2007 ª2007 Elsevier Inc. 955
expression of mGluR5, the major Gp1 mGluR in the fore-
brain. By crossing two mutant lines, the functional relation-
ship between two protein products can be examined;
genetic ‘‘rescue’’ occurs when single mutant phenotypes
are attenuated in the double mutant. The power of this
approach in the murine model is two-fold: (1) it is a precise
and selective method to reduce mGluR5 function, and (2) it
permits analysis of diverse phenotypes across many de-
velopmental time points, using a variety of experimental
methods both in vitro and in vivo. In addition, unlike simpler
genetically modifiable organisms, endophenotypes identi-
fied in this mammalian model not only can serve to estab-
lish genetic interaction, but also may bear direct relation to
the phenotype in humans with the disease.
Fmr1 mutant mice (The Dutch-Belgian Fragile X Consor-
tium, 1994) were crossed with Grm5 mutant mice (Lu et al.,
1997) to produce Fmr1 knockout animals with a selective
reduction in mGluR5 expression (Figure S2). To increase
the therapeutic relevance, we concentrated on animals
with a 50% reduction in mGluR5 rather than a complete
knockout (which impairs brain function [Jia et al., 1998;
Lu et al., 1997]). Littermates with four different genotypes
were created in our cross: wild-type [Fmr1 (+/Y) Grm5
(+/+)], Fmr1 knockout [Fmr1 (/Y) Grm5 (+/+)], Grm5 het-
erozygote [Fmr1 (+/Y) Grm5 (+/)], and the knockout/het-
erozygote cross [Fmr1 (/Y) Grm5 (+/)]; these animals
are termed WT, KO, HT, and CR, respectively. In all cross-
ings, animals were on the C57Bl/6J clonal background.
The key question that we address in this study is if a
reduction of mGluR5 expression will correct diverse fragile
X mutant phenotypes, as predicted by the mGluR theory
(Figure S1). Our genetic rescue strategy rests on the as-
sumption that the FMRP-regulated ‘readout’’ of mGluR5
activation is modulated by Grm5 gene dosage. One
FMRP-regulated consequence of mGluR5 activation is
hippocampal long-term synaptic depression (LTD), which
is approximately doubled in the KO (Huber et al., 2002). It
had already been established that there is a significant
effect of mGluR5 expression level on LTD in the C57Bl/
6J WT background (Huber et al., 2001), and we confirmed
in the present study that a 50% reduction in mGluR5 pro-
tein expression also significantly reduces LTD in the Fmr1
KO background (Figure S2). We therefore went on to
examine diverse phenotypes with relevance to the human
disorder, including experience-dependent cortical devel-
opment, hippocampus-dependent memory, altered
body growth, seizure, and postpubertal macroorchidism.
All analyses of these mice were performed ‘blind,’’ with-
out experimenter knowledge of the genotype. Note that,
in each experiment, three outcomes were possible: the re-
duced Grm5 gene dosage could ameliorate, exacerbate,
or have no effect on Fmr1 mutant phenotypes.
Altered Ocular Dominance Plasticity in Fmr1 KO
Mice Is Rescued by Reducing mGluR5 Expression
Ocular dominance (OD) plasticity in visual cortex, elicited by
temporary monocular deprivation (MD), is the classic exam-
ple of how experience modifies the brain during critical pe-
riods of development. Here, we use this paradigm to study
theinteraction of genesandenvironment in a diseasemodel.
Visually evoked potentials (VEPs) were recorded in the
visual cortex of awake mice (Figure 1A), as described pre-
viously (Frenkel and Bear, 2004). We initially assessed
absolute levels of visual responsiveness across genotypes
on postnatal day (P) 28 and found no difference (Figure 1B).
Additional mice were studied before and after MD begun
on P28. Previous studies using the chronic VEP method
have shown how visual responses evolve during the
course of MD (Figure S3). Closure of the contralateral eye-
lid initially causes depression of responses to the deprived
(contralateral)-eye (apparent at 3 days MD), followed by
potentiation of nondeprived (ipsilateral)-eye responses
(apparent by 7 days MD) (Frenkel and Bear, 2004). Be-
cause they are recorded chronically, changes in VEPs for
each animal can be conveniently described by two values:
the fractional change from baseline in contralateral-eye
response, and the fractional change from baseline in the
ipsilateral-eye response. For reference, average effects
(±SEM) of 3 and 7 days of MD in WT mice from a previous
study (Frenkel and Bear, 2004) appear in Figure S3.
In the current study we also found that the response to
3 days MD in WT mice was dominated by deprived-eye
depression, as expected. In KO littermates, however, the
response to brief MD was characterized by substantial
open-eye potentiation, reminiscent of what happens in
WT mice after longer periods of MD. On the other hand,
the HT mice showed a ‘hypoplastic’ response to MD,
as they lacked significant deprived-eye depression. How-
ever, crossing the two mutant mice resulted in a pheno-
type very similar to WT that was again dominated by
deprived-eye depression (Figure 1C).
Plots of the average (±SEM) fractional changes after
3 days MD in the four genotypes are shown in Figure 1D.
The KO mice displayed increased plasticity compared to
the WT (MANOVA WT:KO, p = 0.011); HT mice displayed
diminished plasticity compared to WT (MANOVA WT:HT,
p = 0.013); CR mice showed a rescue of the KO phenotype
and were not significantly different from WT (MANOVA
WT:CR, p = 0.8268, KO:CR p = 0.037, HT:CRS p = 0.161).
Since the KO and HT mutations affected OD plasticity in
opposite directions, one could question whether the CR
phenotype reflects rescue or the simple addition of two
independent effects. However, a compound phenotype
would be the absence of deprived-eye depression (the ef-
fect of reducing mGluR5) and an exaggeration of open-
eye potentiation (the effect of reducing FMRP). Instead,
we observe a phenotype in the CR mice that is signifi-
cantly different from KO mice, and not significantly differ-
ent from WT. Thus, reducing mGluR5 by 50% corrects the
defect in plasticity caused by the absence of FMRP.
Density of Dendritic Spines on Cortical Pyramidal
Neurons Is Increased in Fmr1 KO and Rescued by
Reducing mGluR5 Expression
Abnormalities in dendritic spines, the major targets of ex-
citatory synapses in the brain, have long been associated
956 Neuron 56, 955–962, December 20, 2007 ª2007 Elsevier Inc.
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Correction of Fragile X Syndrome in Mice
with various forms of human mental retardation, including
FXS. The increased spine density phenotype observed in
humans has been recapitulated in the Fmr1 KO mouse
(reviewed by Grossman et al. [2006]). Because one protein
synthesis-dependent consequence of activating Gp1
mGluRs on cortical neurons in vitro is an increase in the
density of long, thin spines (Vanderklish and Edelman,
2002), we hypothesized that FMRP and mGluR5 antago-
nistically regulate dendritic spine density in vivo.
We chose to examine this question in layer 3 pyramidal
neurons of binocular visual cortex at P30, since we had
established that OD plasticity at this age was altered in
the Fmr1 KO mice. Dendritic spine density was analyzed
separately in apical and basal branches across the four
genotypes, using the Golgi-Cox silver staining method
(Figure 2A). We observed a highly significant increase in
total dendritic spine density in the KO, readily apparent
as a rightward shift in the cumulative probability histogram
(Figure 2B). Reducing mGluR5 expression had no effect
on spine density in the HT mice, but the fragile X pheno-
type was completely rescued in the CR mice (apical
Kruskal-Wallis test, p < 0.0001; Kolmogorov-Smirnov
test, WT:KO p < 0.0001, WT:HT p = 0.3920, CR:WT p =
0.4407, CR:KO p < 0.0001; basal Kruskal-Wallis test, p <
0.0001; Kolmogorov-Smirnov test WT:KO p < 0.0001,
WT:HT p > 0.9999, CR:WT p > 0.9999, CR:KO p < 0.0001).
We also performed a segmental analysis of spine den-
sity across the four genotypes. Consistent with previous
observations, we observed an inverted U-shaped distribu-
tion of synapses in both apical and basal branches across
all genotypes. However, as shown in Figure 2C, the den-
sity of spines was uniformly increased in the Fmr1 KO
and rescued in the CR (repeated-measures ANOVA:
apical distance p < 0.0001, apical distance 3 genotype
p < 0.0001, apical genotype p < 0.0001, basal distance
p < 0.0001, basal distance 3 genotype p = 0.0181, basal
genotype p < 0.0001; ANOVA genotype: apical, basal, 10–
100, in 10 mm segments p < 0.0001; unpaired Student’s t
tests apical, basal, 10–100, in 10 mm segments WT:KO p <
0.05, WT:HT p > 0.05, WT:CR p > 0.05, KO:CR p < 0.05).
These results suggest that neither the Fmr1 KO phenotype
nor the rescue by selective reduction in gene dosage in
the CR reflects a redistribution of synapses within the
segment.
Increased Basal Protein Synthesis
in Hippocampus of Fmr1 KO Mice Is
Rescued by Reducing mGluR5 Expression
A previous study reported an elevated basal rate of in vivo
protein synthesis in the hippocampus of Fmr1 KO mice
(Qin et al., 2005). We asked if this difference could also
be observed in hippocampal slices in vitro by examining
the incorporation of
35
S-methionine/cysteine into new
protein. We observed a significant effect of genotype on
protein synthesis (Figure 3A). The increased protein syn-
thesis seen in KO hippocampus was prevented by selec-
tive reduction in mGluR5 gene dosage.
Electrophoretic separation of radiolabeled translation
products (Figure 3B) suggests that increased protein syn-
thesis in the KO is not limited to one or few predominant
protein species but rather extends across a broad range
Figure 1. Genetic Rescue of OD Plasticity Phenotype in FXS
(A) Schematic of the mouse visual pathway and position of the record-
ing electrode in primary visual cortex.
(B) Absolute VEP amplitudes recorded during binocular viewing across
contrasts (0–100%, square reversing at 1 Hz, 0.05 cycles/degree).
No significant differences across genotypes (n = 46 WT, n = 33 KO,
n = 8 HT, n = 20 CR hemispheres, MANOVA p = 0.0868).
(C) Effect of 3 day MD on VEP amplitude (data expressed as
mean ± SEM, normalized to day 0 ipsilateral eye value. (C
1
) WT mice
(n = 19). Note significant deprived eye depression. (C
2
) KO mice
(n = 18). Note significant open eye potentiation. (C
3
) HT mice (n = 16).
Note absence of deprived eye depression. (C
4
) CR mice (n = 13).
Note rescue of KO phenotype. Post hoc Student’s t tests: *Significantly
different from baseline (day 0).
(D) Plots (mean ± SEM) of the fractional change in open and deprived eye
responses after 3 day MD show rescue of the KO phenotype in CR mice.
Neuron 56, 955–962, December 20, 2007 ª2007 Elsevier Inc. 957
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Correction of Fragile X Syndrome in Mice
of resolved molecular weights. Because the rate of protein
synthesis was unaffected in the HT mice relative to WT,
the rescue in the CR mice is unambiguous and does not
simply reflect an offsetting decrease in synthesis of a
separate pool of proteins.
Inhibitory Avoidance Extinction Is Exaggerated in
Fmr1 KO Mice and Rescued by Reducing
mGluR5 Expression
Although humans with FXS show mental retardation in the
moderate to severe range, prior studies of cognitive per-
formance in Fmr1 KO mice on the C57Bl/6J clonal back-
ground have revealed only subtle deficits (Bernardet and
Crusio, 2006). Consistent with these observations, we
found that acquisition of one-trial inhibitory avoidance
(IA), a hippocampus-dependent memory, did not differ
from normal in the Fmr1 KO mice. However, we were
inspired to additionally investigate IA extinction (IAE) by
a recent report that this process requires protein synthesis
in the hippocampus (Power et al., 2006). We discovered
that IAE is exaggerated in the Fmr1 KO mouse and that
this phenotype is corrected by reducing expression of
mGluR5.
Adult mice of all four genotypes were given IA training,
followed at 6 and 24 hr by IAE training (Figure 4A). For
each animal, we measured the latency to enter the dark
side of the box on the first trial (baseline), the latency
6 hr later (postacquisition) to assess IA memory, and again
at 24 and 48 hr (post-extinction 1 and 2, respectively) to
assess IAE. As shown in Figure 4B, animals of all four
genotypes showed both significant IA acquisition at 6 hr
and extinction by 48 hr. A global statistical test suggested
that the pattern of learning across time varied across ge-
notypes (repeated-measures ANOVA genotype 3 time
p = 0.0239). As shown in Figures 4C–4E, these differences
are likely due to extinction rather than acquisition of inhib-
itory avoidance. At 6 hr, there was no difference across
genotypes in latency to enter (6 hr ANOVA p = 0.1525);
however, at 24 hr KO mice showed significantly shorter
latencies, suggesting exaggerated extinction in the ab-
sence of FMRP. This phenotype was rescued by selec-
tive reduction in mGluR5 gene dosage in the CR mice
(24 hr ANOVA p = 0.0013; Student’s t tests: WT:KO
Figure 2. Genetic Rescue of Dendritic Spine Phenotype in
FXS
(A) Representative images from apical (A
1
) and basal (A
2
) dendritic seg-
ments of layer 3 pyramidal neurons in the binocular region of primary
visual cortex of all four genotypes collected at P30.
(B) Cumulative percent spines per mm in each dendritic segment; api-
cal branches, B
1
; basal branches, B
2
(n = 80 WT, 80 KO, 60 HT, 80 CR
apical and basal branches, respectively). (C) Segmental analysis of
spine density; number of spines per 10 mm bin, given as distance
from the origin of the branch, for apical (C
1
) and basal (C
2
) segments
across four genotypes.
Figure 3. Genetic Rescue of Protein Synthesis Phenotype in
FXS
(A) Significant differences in the levels of protein synthesis exist across
genotypes in the ventral hippocampus (n = 10 samples, 5 animals per
genotype). KO mice showed increased protein synthesis (mean ±
SEM: WT 389 ± 33.77 cpm/mg; KO 476 ± 29.98 cpm/mg; post hoc
paired Student’s t test WT:KO p = 0.004). Protein synthesis levels in
the HT mice were no different than WT (HT 409 ± 42.99 cpm/mg).
Increased protein synthesis seen in the KO were rescued in the CR
mice (CR 374 ± 50.81 cpm/mg). Post hoc paired Student’s t tests:
*significantly different from WT,
y
significantly different from KO.
(B) Representative autoradiogram shows that synthesis of many
protein species is elevated in the KO compared to all other genotypes.
958 Neuron 56, 955–962, December 20, 2007 ª2007 Elsevier Inc.
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Correction of Fragile X Syndrome in Mice
p < 0.0001,WT:HT p = 0.8251, WT:CR p = 0.1156, KO:CR
p = 0.0132).
Because the primary aim of this study is to examine
genetic interaction between Fmr1 and Grm5, we also per-
formed a multivariate analysis that takes into consider-
ation both acquisition and extinction as they vary across
genotypes. As shown in Figure 4F, KO animals showed
significant difference in 24 hr latency (memory retention)
as it varied with 6 hr latency (memory acquisition), and
this difference was rescued by the selective reduction in
mGluR5 gene dosage in the CR mice (MANOVA for geno-
type 6:24 p = 0.0054, MANOVA WT:KO p = 0.0005, WT:HT
0.0785, KO:CR p = 0.0490, WT:CR p = 0.1863). The differ-
ence in retention across all genotypes was not significant
by 48 hr. Regardless of whether this KO phenotype
reflects exaggerated extinction or diminished stability of
the formed memory, there clearly is a significant genetic
interaction between Fmr1 and Grm5: the Fmr1 KO pheno-
type is rescued by the selective reduction in mGluR5
expression.
Audiogenic Seizures and Accelerated Body
Growth in Fmr1 KO Mice Are Rescued
by Reducing mGluR5 Expression
Consistent with neurological findings in fragile X patients,
previous studies have shown increased seizure suscepti-
bility in the Fmr1 KO mouse, using both in vitro and in vivo
epilepsy models (Bernardet and Crusio, 2006). We em-
ployed the audiogenic seizure (AGS) paradigm, which
shows a robust phenotype in Fmr1 KO mice and exhibits
developmental changes consistent with epilepsy in
human FXS (Yan et al., 2005). Because C57Bl/6 WT
mice are normally seizure resistant (Robertson, 1980), sei-
zures in the KO mice are a specific consequence of the
absence of FMRP. As shown in Table S1, significant differ-
ences in AGS susceptibility were observed across the four
genotypes examined. WT and HT mice showed zero inci-
dences of AGS, as expected, whereas 72% of the KO
mice had a seizure in response to the tone (Mann-Whitney
U test WT:KO p < 0.0001). This mutant phenotype was sig-
nificantly attenuated in the CR mice (Mann-Whitney U test
CR:KO p = 0.028). Thus, chronic reduction of mGluR5
gene dosage in KO mice produced a substantial rescue
of the seizure phenotype that is caused specifically by
the lack of FMRP.
Children with FXS show accelerated prepubescent
growth (Loesch et al., 1995). We discovered that this phe-
notype is recapitulated in the KO mouse and is rescued by
reducing mGluR5 gene dosage (Figure S4). At weaning
(P20–21), animals from all four genotypes had similar
body weights, but by P26 KO mice showed a slight
(10%) but significant increase in body weight as com-
pared to WT animals at the same age. This difference
was not observed in either the HT or CR mice (ANOVA
p = 0.048, post hoc Student’s t tests WT:KO p = 0.017,
KO:CR p = 0.004, CR:WT p = 0.818). The WT:KO body
weight difference was maximal at P30 (15%), when it
was again rescued by a reduction in mGluR5 gene dosage
in the CR mice (ANOVA p = 0.005, post hoc Student’s
t tests WT:KO p = 0.020, KO:CR p = 0.001, CR:WT
p = 0.555). As in humans, the KO growth increase in
mice was no longer apparent after adolescence (P45).
Figure 4. Genetic Rescue of Behavioral
Learning and Memory Phenotype in FXS
(A) Experimental design. Animals of all four
genotypes (n = 15 WT, n = 15 KO, n = 20 HT,
n = 17 CR) were given IA training at time 0,
and the latency to enter the dark side was mea-
sured at 6 hr. They were then given IAE training,
and latency was again measured at 24 hr. Test-
ing was followed by another round of IAE train-
ing, and latency was measured again at 48 hr.
(B) Animals in all four genotypes showed signif-
icant acquisition and extinction. Post hoc Stu-
dent’s t tests: *significantly different from time
0 hr,
y
significantly different from time 6 hr.
(C) Raw data for acquisition of IA in the four
genotypes. Each line represents the change
in latency to enter the dark side for one mouse
(data from some mice superimpose).
(D) Raw data for extinction 1 in the four geno-
types.
(E) Comparison of latency (mean ± SEM)
across genotypes for 6 hr time point (postac-
quisition) and 24 hr time point (post-extinction
1). Post hoc Student’s t tests: *significantly dif-
ferent from WT,
y
significantly different from KO.
(F) Multivariate analysis of extinction as a func-
tion of acquisition at 24 hr time point (post-
extinction 1) and 48 hr time point. Plotted are
mean latencies ± SEM.
Neuron 56, 955–962, December 20, 2007 ª2007 Elsevier Inc. 959
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Correction of Fragile X Syndrome in Mice
Macroorchidism in Fmr1 KO Mice Is Not Rescued
by Reducing mGluR5 Expression
Children with FXS (and KO mice) have dysmorphic fea-
tures, including postadolescent macroorchidism. Testes
express Gp1 mGluRs (Storto et al., 2001), so we won-
dered if this phenotype might also be rescued in our CR
mice. Postadolescent testicular weight was increased by
24% in KO mice compared to WT (p < 0.0004; Student’s
t test); however, there was no rescue of this phenotype
in the CR mice (Figure S5). To investigate if the absence
of rescue was a matter of gene dosage, we generated
KO mice that had a complete absence of mGluR5 [Fmr1
(/Y) Grm5 (/), dKO]. Again, however, there was no
rescue of the testicular phenotype.
DISCUSSION
The goal of this study was to test a key prediction of the
mGluR theory—that aspects of FXS can be corrected by
downregulating signaling through group 1 mGluRs (Bear
et al., 2004). Each analysis was designed to examine a dif-
ferent dimension of the disorder in mice with relevance to
the human syndrome, ranging from the cognitive to the
somatic. The experiments assayed dysfunction in very
different neural circuits; and for each, three outcomes
were possible: amelioration, exacerbation, or persistence
of Fmr1 mutant phenotypes in mice with reduced expres-
sion of mGluR5. Thus, it is remarkable that by reducing
mGluR5 gene dosage by 50%, we were able to bring mul-
tiple, widely varied fragile X phenotypes significantly
closer to normal.
A novel aspect of the current study was the use of OD
plasticity as an in vivo assay of how experience-dependent
synaptic modification is altered by the loss of FMRP. MD
sets in motion a sequence of synaptic changes in visual
cortex, characterized by a rapid and persistent loss of re-
sponsiveness to the deprived eye and a slower compensa-
tory increase in responsiveness to the nondeprived eye
(Frenkel and Bear, 2004). Because MD triggers mecha-
nisms of synaptic depression and potentiation, as well as
homeostatic adaptations to an altered environment, OD
plasticity is a particularly rich paradigm for understanding
the interactions of genes and experience. The intersecting
trends of using mice to study OD plasticity mechanisms
and to model human diseases provided the opportunity
to use this paradigm to get a more precise understanding
of how development goes awry in a genetic disorder.
Previous work suggested that Gp1 mGluR signaling is
highest in visual cortex during the period of maximal syn-
aptic plasticity (Dudek and Bear, 1989), and the current
findings strongly suggest an important role for mGluR5
in OD plasticity. Although more experiments will be re-
quired to pinpoint this role, an obvious clue comes from
the genetic interaction with Fmr1. FMRP can act as
a translational suppressor (Brown et al., 2001; Qin et al.,
2005), and OD plasticity, like many forms of persistent
synaptic modification, requires protein synthesis (Taha
and Stryker, 2002). Thus, our findings suggest the intrigu-
ing hypothesis that the rate of plasticity in visual cortex is
determined by the level of activity-dependent protein syn-
thesis, which is stimulated by mGluR5 and inhibited by
FMRP. Consistent with this model, the phenotype in
Fmr1 KO mice appears to reflect ‘hyperplasticity,’ since
3 days of MD yielded effects on VEPs that normally require
7 days. This exaggerated plasticity was corrected by re-
ducing mGluR5 expression by 50%.
Although we observed an increased spine density in the
visual cortex of KO mice, there was no apparent difference
in the amplitude of VEPs at P30. This discrepancy may be
because VEPs were recorded in layer 4, whereas the spine
measurements were made on layer 3 neurons. In any
case, the clear mutant spine phenotype in layer 3 gave
us the opportunity to examine if this structural defect
could also be corrected by decreasing mGluR5. We ob-
served a remarkable rescue of the fragile X spine pheno-
type in the CR mice, despite the fact that reducing
mGluR5 expression in the HT mice had no effect on spine
density. Thus, although the reduction in Grm5 gene dos-
age is not sufficient to alter spine density by itself, it com-
pletely corrects the defect in fragile X mice.
Strain-specific variation has confounded previous at-
tempts to identify a behavioral learning and memory
phenotype in the Fmr1 KO (Bernardet and Crusio, 2006).
Consistent with earlier findings, we were unable to detect
a significant IA deficit in the Fmr1 KO mice on the C57BL/6
background. On the other hand, we were able to detect
a difference in the rate of IA extinction that could be cor-
rected by reducing mGluR5 expression. Because IA in-
duces LTP of Schaffer collateral synapses in area CA1 of
the hippocampus (Whitlock et al., 2006
), it is tempting to
speculate that IA extinction is exaggerated in Fmr1 KO
mice due, at least in part, to excessive mGluR-dependent
synaptic weakening (Huber et al., 2002; Zho et al., 2002).
Unbalanced LTD could account for the cognitive impair-
ment that is the hallmark of fragile X.
Fragile X is a syndromic disorder. In addition to mental
retardation, associated features of the disease in humans
include childhood epilepsy, altered body growth, and
postpubertal macroorchidism. In the case of epilepsy
and macroorchidism, these phenotypes have been reca-
pitulated in the mouse model (Bernardet and Crusio,
2006); however, it was previously unknown that Fmr1
KO mice show a similar disruption in body growth. Both
the body growth and AGS phenotypes were ameliorated
in the CR mice; however, there was no evidence of an in-
teraction between FMRP and mGluR5 in the control of tes-
ticle size. These results argue against a role for mGluR5 in
the pathogenesis of the macroorchidism phenotype in
FXS, but we cannot rule out a contribution of the other
Gp1 mGluR (mGluR1).
Conclusion
Although we studied a range of phenotypes, a simple way
to conceptualize the constellation of findings is that fragile
X is a disorder of excess—excessive sensitivity to environ-
mental change, synaptic connectivity, protein synthesis,
960 Neuron 56, 955–962, December 20, 2007 ª2007 Elsevier Inc.
Neuron
Correction of Fragile X Syndrome in Mice
memory extinction, body growth, and excitability—and
these excesses can be corrected by reducing mGluR5.
Although the precise molecular basis of the interaction
remains to be determined, the data show unambiguously
that mGluR5 and FMRP act as an opponent pair in several
functional contexts and support the theory that many CNS
symptoms in fragile X are accounted for by unbalanced
activation of Gp1 mGluRs. These findings have major ther-
apeutic implications for FXS and autism (see Bear et al.,
2008).
EXPERIMENTAL PROCEDURES
Animals
Fmr1 mutant mice (Jackson Labs) were crossed with Grm5 mutants
(Jackson Labs) to produce mice of four genotypes. In all crossings,
animals were on the C57Bl/6J clonal background. In an effort to reduce
variability due to rearing conditions, all experimental animals were
bred from Fmr1 heterozygote mothers, group housed (animals weaned
to solitary housing were excluded), and maintained in a 12:12 hr light:
dark cycle. Paternal genotype varied between crossings and included
WT, Grm5 HT, or Grm5 KO.
Genotyping
See Figure S2 and the Supplemental Data.
Electrophysiology and Spine Measurements
Transverse hippocampal slices were prepared from P25–30 mice and
mGluR-LTD was studied as described by Huber et al. (2001). VEP
recordings and monocular deprivation were performed as previously
described (Frenkel and Bear, 2004). Spines were analyzed using the
Golgi-Cox method as described by Hayashi et al. (2004). Animal n =
8 WT, 8 KO, 6 HT, 8 CR; dendritic segment n = 80 WT, 80 KO,
60 HT, 80 CR apical and basal branches, respectively. All protrusions,
irrespective of their morphological characteristics, were counted as
spines if they were in direct continuity with the dendritic shaft. In total,
68,032 spines were counted across all four genotypes.
Inhibitory Avoidance Extinction, Metabolic Labeling, and
Audiogenic Seizure
IAE experiments were performed as previously described (Power et al.,
2006). Metabolic labeling experiments were similar to those described
in Raymond et al. (2000). AGS experiments were performed as
described by Yan et al. (2005). See Supplemental Data.
Statistical Analysis
In all cases, post hoc comparisons between genotypes were made
only if global analysis indicated a statistically significant (p < 0.05)
effect of genotype. Outliers (R2 SD from the mean) were excluded.
For AGS experiments, nonparametric statistics (Kruskall-Wallis,
Mann-Whitney U) were used since incidence scores were bimodal
(yes/no). For all other analysis, parametric tests (ANOVA, MANOVA,
two-tailed paired and unpaired Student’s t tests, assuming equal var-
iance) were used. For the metabolic labeling experiments, the post hoc
paired Student’s t test was used to eliminate the variability due to
strength of radioactive label on different experimental days and was
justified by the experimental design (samples were collected with
yoked, rather than randomized, controls).
Supplemental Data
The Supplemental Data for this article can be found online at http://
www.neuron.org/cgi/content/full/56/6/955/DC1/.
ACKNOWLEDGMENTS
We are grateful to M. Shuler for helpful discussion of appropriate
statistical analysis; K. Oram, C. Dudley, A. Topolszki, T. Udaka, and
C. Poo for genotyping and animal care; E. Sklar for technical assis-
tance and construction of AGS stimulus; M. Frenkel for use of previ-
ously published data; S. Meagher for administrative support; A. Kraev
and J. Roder for Grm5 genotyping protocols; K. Huber, K. Wiig, A. Go-
vindarajan, R. Paylor, W. Chen, B. Yoon, Q. Yan, R. Bauchwitz, M.
Tranfaglia, E. Klann, E. Berry-Kravis, and A. Heynen for helpful discus-
sions. Supported by the NIMH, NICHD, National Fragile X Foundation,
FRAXA, Simons Foundation. M.F.B. discloses a financial interest in
Seaside Therapeutics.
Received: March 29, 2007
Revised: October 8, 2007
Accepted: December 3, 2007
Published: December 19, 2007
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    • "In ASD, estimated to affect 1 in 88 children by the Centers for Disease Control (CDC), a meta-analysis of studies revealed a rate of ID as high as 75 % [1]; although more recent large-scale epidemiological studies reflect a rate of approximately 41 %. Recent advances in understanding the molecular mechanisms underlying NDD through animal models [2][3][4][5]have led to targeted controlled trials with pharmacological agents designed to normalize molecular abnormalities, synaptic function, cognition, and behavior in humans with these conditions. FXS, the most common inherited form of ID, is by far the leading example of this translational effort. "
    [Show abstract] [Hide abstract] ABSTRACT: Background Recent advances in understanding molecular and synaptic mechanisms of intellectual disabilities (ID) in fragile X syndrome (FXS) and Down syndrome (DS) through animal models have led to targeted controlled trials with pharmacological agents designed to normalize these underlying mechanisms and improve clinical outcomes. However, several human clinical trials have failed to demonstrate efficacy of these targeted treatments to improve surrogate behavioral endpoints. Because the ultimate index of disease modification in these disorders is amelioration of ID, the validation of cognitive measures for tracking treatment response is essential. Here, we present preliminary research to validate the National Institutes of Health Toolbox Cognitive Battery (NIH-TCB) for ID. Methods We completed three pilot studies of patients with FXS (total n = 63; mean age 19.3 ± 8.3 years, mean mental age 5.3 ± 1.6 years), DS (n = 47; mean age 16.1 ± 6.2, mean mental age 5.4 ± 2.0), and idiopathic ID (IID; n = 16; mean age 16.1 ± 5.0, mean mental age 6.6 ± 2.3) measuring processing speed, executive function, episodic memory, word/letter reading, receptive vocabulary, and working memory using the web-based NIH-TB-CB, addressing feasibility, test-retest reliability, construct validity, ecological validity, and syndrome differences and profiles. ResultsFeasibility was good to excellent (≥80 % of participants with valid scores) for above mental age 4 years for all tests except list sorting (working memory). Test-retest stability was good to excellent, and convergent validity was similar to or better than results obtained from typically developing children in the normal sample for executive function and language measures. Examination of ecological validity revealed moderate to very strong correlations between the NIH-TCB composite and adaptive behavior and full-scale IQ measures. Syndrome/group comparisons demonstrated significant deficits for the FXS and DS groups relative to IID on attention and inhibitory control, a significant reading weakness for FXS, and a receptive vocabulary weakness for DS. Conclusions The NIH-TCB has potential for assessing important dimensions of cognition in persons with ID, and several tests may be useful for tracking response to intervention. However, more extensive psychometric studies, evaluation of the NIH-TCB’s sensitivity to change, both developmentally and in the context of treatment, and perhaps establishing links to brain function in these populations, are required to determine the true utility of the battery as a set of outcome measures.
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    • "The absence of FMRP might also induce an increase in the translation of proteins involved in internalization of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (ionotropic glutamate receptors), which could lead to elongation of the dendritic spine and fewer glutamate ionotropic receptors on the post-synaptic membrane (Figure 2). Fmr1 knockout mice, heterozygous for mGlur5 (50% reduction of expression of this receptor) showed a rescue of the synaptic phenotype [33]. AGG triplets may be interspersed every 9–10 CGGs [17]. "
    [Show abstract] [Hide abstract] ABSTRACT: Fragile X syndrome (FXS) is the most common cause of inherited intellectual disability, caused by CGG expansion over 200 repeats (full mutation, FM) at the 5' untranslated region (UTR) of the fragile X mental retardation 1 (FMR1) gene and subsequent DNA methylation of the promoter region, accompanied by additional epigenetic histone modifications that result in a block of transcription and absence of the fragile X mental retardation protein (FMRP). The lack of FMRP, involved in multiple aspects of mRNA metabolism in the brain, is thought to be the direct cause of the FXS phenotype. Restoration of FMR1 transcription and FMRP production can be obtained in vitro by treating FXS lymphoblastoid cell lines with the demethylating agent 5-azadeoxycytidine, demonstrating that DNA methylation is key to FMR1 inactivation. This concept is strengthened by the existence of rare male carriers of a FM, who are unable to methylate the FMR1 promoter. These individuals produce limited amounts of FMRP and are of normal intelligence. Their inability to methylate the FMR1 promoter, whose cause is not yet fully elucidated, rescues them from manifesting the FXS. These observations demonstrate that a therapeutic approach to FXS based on the pharmacological reactivation of the FMR1 gene is conceptually tenable and worthy of being further pursued.
    Full-text · Article · Aug 2016
    • "Our present studies are also relevant to the mGluR hypothesis of autism, which proposes that dysregulation of mGluR function is a critical neuropathology in ASDs (Bear et al., 2004). Abnormalities in mGlu5 signaling in the hippocampus have been reported in several models of ASDs, in particular the FMR1 KO model and mGlu5 antagonist reverse molecular and behavioral deficits in FMR1 knockout mice, and have also been tested in clinical trials for fragile-X patients, with some promising results (Dölen et al., 2007 Jacquemont et al., 2011). Our data demonstrate that GluD1 is an important regulator of mGlu5 signaling and protein synthesis, and further analysis of the roles of GluD1 is necessary to fully understand its contribution to central nervous system physiology and neuropsychiatric disorders. "
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