# Mouse models of the fragile X premutation and fragile X-associated tremor/ataxia syndrome

Robert F Berman, Ronald Am Buijsen, Karen Usdin, Elizabeth Pintado, Frank Kooy, Dalyir Pretto, Isaac N Pessah, David L Nelson, Zachary Zalewski, Nicolas Charlet-Bergeurand, et al.

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Robert F Berman, Ronald Am Buijsen, Karen Usdin, Elizabeth Pintado, Frank Kooy, et al.. Mouse models of the fragile X premutation and fragile X-associated tremor/ataxia syndrome. Journal of Neurodevelopmental Disorders, 2014, 6 (1), pp.25. 10.1186/1866-1955-6-25. hal-05013743

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# Mouse models of the fragile X premutation and fragile X-associated tremor/ataxia syndrome

Robert F Berman¹*, Ronald AM Buijsen², Karen Usdin³, Elizabeth Pintado⁴, Frank Kooy⁵, Dalyir Pretto⁶, Isaac N Pessah⁷, David L Nelson⁸, Zachary Zalewski⁸, Nicholas Charlet-Bergeurand⁹, Rob Willemsen² and Renate K Hukema²

**Abstract**

Carriers of the fragile X premutation (FPM) have CGG trinucleotide repeat expansions of between 55 and 200 in the 5'-UTR of *FMR1*, compared to a CGG repeat length of between 5 and 54 for the general population. Carriers were once thought to be without symptoms, but it is now recognized that they can develop a variety of early neurological symptoms as well as being at risk for developing the late onset neurodegenerative disorder fragile X-associated tremor/ataxia syndrome (FXTAS). Several mouse models have contributed to our understanding of FPM and FXTAS, and findings from studies using these models are summarized here. This review also discusses how this information is improving our understanding of the molecular and cellular abnormalities that contribute to neurobehavioral features seen in some FPM carriers and in patients with FXTAS. Mouse models show much of the pathology seen in FPM carriers and in individuals with FXTAS, including the presence of elevated levels of *Fmr1* mRNA, decreased levels of fragile X mental retardation protein, and ubiquitin-positive intranuclear inclusions. Abnormalities in dendritic spine morphology in several brain regions are associated with neurocognitive deficits in spatial and temporal memory processes, impaired motor performance, and altered anxiety. *In vitro* studies have identified altered dendritic and synaptic architecture associated with abnormal Ca²⁺ dynamics and electrical network activity. FPM mice have been particularly useful in understanding the roles of *Fmr1* mRNA, fragile X mental retardation protein, and translation of a potentially toxic polyglycine peptide in pathology. Finally, the potential for using these and emerging mouse models for preclinical development of therapies to improve neurological function in FXTAS is considered.

**Keywords:** CGG trinucleotide repeat, *FMR1*, FMRP, Fragile X premutation, FXTAS, Intranuclear inclusions, Mouse models, RNA toxicity

**Introduction**

The Fragile X mental retardation 1 gene (*FMR1*) is located on the long arm of the X-chromosome at Xq27.3 and codes for the fragile X mental retardation protein (FMRP), which is necessary for normal brain development and synaptic plasticity [1-5]. The fragile X gene carries a variable number of CGG repeats in the 5'-UTR of between 5 and 55 in most individuals (modal value 32 to 33; Figure 1). However, due to instability of the repeat across generations, there are large numbers of individuals who carry an expanded CGG repeat of between 55 and 200. These

individuals are referred to as fragile X premutation (FPM) carriers, and occur in the general population with an estimated frequency of 1 in 209 females and 1 in 430 males [6,7]. Further expansion of the CGG repeat to greater than 200 in the offspring of FPM carriers leads to the full mutation, silencing of *FMR1* expression and fragile X syndrome (FXS), the major known inherited cause of intellectual disability [4,8].

Carriers with 55 to 200 CGG repeats were originally thought to be clinically unaffected. However, it is now known that they can develop a variety of neurological symptoms, including memory problems, deficits in executive function, depression, anxiety, and problems with numerical processing and magnitude estimates [3,10,11]. They are also at risk for developing the late

<footnote>* Correspondence: rfberman@ucdavis.edu
¹Department of Neurological Surgery, Room 502C, UC Davis, 1515 Newton Court, Davis, CA 95618, USA
Full list of author information is available at the end of the article</footnote>
<figure><img src="image_3.png" /><figcaption>**Figure 1** Most individuals in the general population have between 5 and 54 CGG trinucleotide repeats in the 5'-UTR of *FMR1*. Repeat length in the fragile X premutation range is 55 to 200, resulting in an elevation in *FMR1* mRNA levels, a moderate decrease in FMRP and an increased risk of developing FXTAS. Repeat size in the full mutation is >200; *FMR1* transcription is silenced due to DNA hypermethylation, and the absence of FMRP results in fragile X syndrome. (Adapted from [9].) FMRP, fragile X mental retardation protein; FXTAS, fragile X-associated tremor/ataxia syndrome.</figcaption></figure>

onset neurodegenerative disorder fragile X-associated tremor/ataxia syndrome (FXTAS). Major symptoms of FXTAS include tremor, ataxia, impairments in executive function and memory, and cognitive decline and dementia in some patients [12,13]. Neuropathology includes brain atrophy, ventricular enlargement, loss of Purkinje neurons, white matter disease, disruption of nuclear lamin A/C architecture and accumulation of intranuclear protein inclusions [5,14].

The chances of developing FXTAS increase dramatically with age, with approximately 45.5% of male and 16.5% of female FPM carriers over the age of 50 developing FXTAS [15]. Indeed, FXTAS may be one of the more common causes of tremor and ataxia in older adults [16]. Besides age, the risk factors that lead to the development of FXTAS in some, but not all, FPM carriers are unknown, but are likely to include CGG repeat length, additional genetic mechanisms and environmental factors (for example, environmental toxins, other illnesses [3]). Identifying the risk factors for FXTAS is particularly important and animal models will undoubtedly play a major role in this area of research.

Because of the increase in the number of people reaching the age of 65, it is likely that the number of cases of FXTAS will increase accordingly, further highlighting the importance of research on FXTAS [16]. Therefore, it is important to understand the underlying pathology in FXTAS, to establish its developmental time course, and to develop rational treatments to delay or halt progression of disease and improve neurological function.

## Review

### Pathogenesis in affected FPM carriers and in FXTAS

Pathology in affected FPM carriers and in individuals with FXTAS is thought to be the result of RNA toxicity caused by 2- to 8-fold elevated levels of CGG-repeat-bearing *FMR1* mRNA. As depicted in Figure 2A, elevated *Fmr1* mRNA with a CGG repeat expansion is thought to sequester proteins critical for normal cell function, resulting in pathology. This hypothesis is supported by the finding that inclusions isolated from postmortem brain tissue from patients with FXTAS contain *FMR1* mRNA and over 30 proteins, many critical for normal cell function, such as lamin A/C, γH2AX, Sam 68, drosha, Ku86 and hnRNPA2 [17-19]. However, recent findings have suggested an additional model for toxicity, as depicted in Figure 2B, in which a potentially toxic polyglycine-containing peptide is produced as the result of a CGG repeat-mediated non-ATG translation (RAN) mechanism [20]. Research using animal models has provided much of the evidence supporting these theories as presented in this review.

### Development of mouse models

Several mouse models have been developed to study the FPM and FXTAS. These models show much of the pathology associated with CGG repeat expansions on *FMR1*. Table 1 compares pathology seen in FXTAS with that reported in CGG knock-in (KI) mouse models, including molecular, histological and some behavioral deficits. However, no models have been completely successful in
<figure><img src="image_4.png" /></figure>

**Figure 2** Potential mechanisms of CGG-repeat RNA toxicity in FMP carriers. (A) Protein sequestration model: RNA binding proteins are sequestered through their interactions with the expanded CGG-repeat RNA. These proteins can in turn recruit other proteins. The net result of sequestration of these proteins is that they are unavailable to carry out their normal functions and critical cellular processes are thereby altered or blocked. (? -Sequestration of SAM68 by CGG expanded repeats is indirect, presumably through protein-protein interactions). (B) Toxic polypeptide model: The 43S translation initiation complex stalls near the CGG repeat hairpin formed on the *FMR1* RNA. This promotes the repeat-associated non-AUG translation of *FMR1* mRNA using a near-AUG start site. This results in a frame shift and the production of polyglycine- and/or polyalanine-containing polypeptides that somehow interfere with normal cell function or may be directly toxic. FMRP, FMRP, fragile X mental retardation protein; ORF, open reading frame; polyA, polyalanine; polyG, polyglycine.

reproducing all of the features reported in affected FPM or individuals with FXTAS. An important example is the absence of any reports of obvious tremor in current mouse models, a defining neurological feature of FXTAS. Therefore, it is acknowledged at the outset that current mouse models only partially recapitulate features of the FPM and FXTAS. The mouse models described below have been developed to study specific aspects of disease associated with CGG repeat expansions; each offers advantages and limitations, and each has already provided important insights into disease mechanisms.

### The Dutch mouse

The study of FXS and FXTAS has been greatly facilitated by the development of animal models that mimic much of the pathology associated with these disorders. The

first mouse model of FXTAS and the FPM was a CGG KI mouse model from the Willemsen laboratory in the Netherlands, the so-called Dutchmouse (CGG<sub>dut</sub> KI). This mouse model was generated by replacing the native murine CGG repeat eight trinucleotides in length (CGG8) within the endogenous *Fmr1* gene with a human CGG98 repeat by homologous recombination in embryonic stem cells [22]. Importantly, while minimal changes to the murine *Fmr1* promoter were made when the targeting construct containing the human (CGG)<sub>98</sub> repeat was generated, the region flanking the repeat in the human *FMR1* was included. These CGG KI mice show moderate instability of repeat length upon paternal and maternal transmission, with both small expansions and contractions (that is, typically fewer than 10 repeats) [22-24]. These CGG<sub>dut</sub> KI mice have been bred onto a C57BL/6 J background over

<table><caption>**Table 1** FXTAS compared to the CGG knock-in mouse model</caption><thead><tr><td>Pathology</td><td>Human FXTAS</td><td>CGG knock-in mouse</td></tr></thead><tbody><tr><td>CGG trinucleotide repeat length</td><td>55 to 199 CGG repeat length, repeat instability</td><td>70 to 300 CGG repeats, modest repeat instability</td></tr><tr><td>Elevated FMR1 mRNA expression</td><td>Increased 2- to 8-fold</td><td>Increased 1.5- to 3-fold</td></tr><tr><td>Fragile X mental retardation protein levels</td><td>Reduced in several brain regions</td><td>Reduced in several brain regions</td></tr><tr><td>Motor impairments</td><td>Tremor/ataxia, postural sway, parkinsonism</td><td>Impaired on rotarod and ladder rung task</td></tr><tr><td>Cognitive Impairments</td><td>Poor working memory, anxiety, depression, social phobia</td><td>Spatial memory deficits, altered anxiety-like behaviors</td></tr><tr><td>Intranuclear inclusions</td><td>Neurons and astrocytes, highly correlated with CGG repeat length, frequency increases with age</td><td>Neurons and astrocytes, related to length of CGG repeat, frequency increases with age</td></tr></tbody></table>

Adapted from [21].
several generations to establish lines with expanded alleles ranging from 70 to greater than 300 CGG repeats [21,22]. Although expected, based on silencing of *FMR1* expression in FXS, no increased methylation of the *Fmr1* gene has been found even with longer CGG repeat expansions (for example, >300). As described below, these mice models exhibit much of the pathology seen in affected FPM carriers and in FXTAS, including increased expression of *Fmr1* mRNA, decreased FMRP, ubiquitin-positive intranuclear inclusions (Figure 3) and evidence for motor and spatial processing deficits [21].

## The National Institutes of Health mouse

A second KI mouse was developed at the National Institutes of Health with an initial CGG118 tract [26,27]. The CGG*nih* KI mice were generated using a different strategy from the CGG*dut* mice. They were developed using a targeting construct in which exon 1 of the mouse gene was retrofitted with two adjacent but incompatible *Sfi* I sites. The repeats were generated *in vitro* in such a way that they were flanked by the appropriate *Sfi* I sites. This allowed the CGG repeats to be inserted into the mouse locus in the correct orientation and in such a way as to make minimal changes to the mouse flanking sequence. As a result of this strategy, the CGG*nih* mouse retains the translational TAA stop codon just upstream of the CGG118 repeat that is present in the endogenous murine gene but not the human gene. As with the CGG*dut* mice, the CGG*nih* mice show elevated *Fmr1* mRNA levels, decreased FMRP levels, moderate intergenerational expansions, no methylation (even when repeat

numbers were >300) and ubiquitin-positive intranuclear inclusions [26].

## Similarities and differences between CGG*dut* and CGG*nih* knock-in models

The two CGG KI mouse models show similarities as well as some differences [26,28]. Both models show several-fold increases in levels of *Fmr1* mRNA and a reduction in brain levels of FMRP that is inversely related to CGG repeat length. However, they differ in that the reduction in FMRP in the CGG*dut* KI mouse (20% to 30%) is typically much less than that reported in the CGG*nih* KI (>50%). Ubiquitin-positive intranuclear inclusions are found in both models, but are more common in neurons and astrocytes in the CGG*dut* KI model [20]. Inclusions in CGG*dut* KI mice are widespread in the brain, including the hippocampus, cortex, cerebellum, olfactory bulb, superior and inferior colliculi, and hypothalamus [24]. Purkinje cell loss is seen in postmortem tissue from FXTAS brains, as well as in the CGG*nih* KI mouse, but has not been reported in the CGG*dut* KI mouse [26]. Behaviorally, there is evidence for memory impairment in both models [29,30], but the CGG*dut* KI mouse shows increased anxiety [31] whereas the CGG*nih* KI mouse shows decreased anxiety [30]. Both models show modest intergenerational repeat instability. Neither model, however, reliably shows large expansions in the length of the CGG repeat tract seen with maternal transmission in FXS, and no methylation or silencing of *Fmr1* expression has been reported in either model. This difference between humans and mice in the frequency of large germline expansions may be due to differences in the length of the perigametic interval in males of both

<figure><img src="image_5.png" /><figcaption>**Figure 3** Ubiquitin-positive intranuclear inclusions in neurons and astrocytes of CGG*dut* knock-in mice. White arrows point to red punctate intranuclear inclusions in pyramidal neurons in motor cortex (A), cortical astrocytes (B) and Bergmann glia in cerebellum (C). Intranuclear inclusions (red) were labeled by immunofluorescence for ubiquitin, neurons (green) for Kv2.1 potassium channels, and astrocytes and Bergmann glia (green) for GFAP. In (B), note an intranuclear inclusion in an adjacent neuron (arrowhead). Nuclei were stained with DAPI. (Adapted from [25]).</figcaption></figure>
to controls, and this impairment was not age-related, as
similar impairment was seen in 20- and 40-week-old
mice. These results provide evidence that CGG repeat
mRNA expression is sufficient to cause Purkinje
neuron dysfunction and loss similar to that reported in
FXTAS [35].

Neuropathological observations to date demonstrate a
connection between formation of intranuclear inclusions
and cell death. While it is tempting to speculate that the
formation of inclusions is the cause of cell loss, such a
conclusion is contingent upon understanding what the
functional ramifications are when proteins and their inter-
acting partners are sequestered within an inclusion body.
A *Drosophila* model ectopically expressing premutation-
length CGG repeats showed a neurodegenerative eye
phenotype and *Hsp70/ubiquitin*-positive inclusions [36].
A subsequent genetic screen showed that CELF1
(CUGBP1), when ectopically expressed, was able to sup-
press the neurodegenerative eye phenotype [37]. CELF1
was also shown to directly interact with hnRNPA2/B1,
known to be present in inclusions of patients with FXTAS
[18]. CELF1 is up-regulated overall in the presence of CUG
repeats >50, contributing to the mis-regulation of mRNA
splicing and translation and the muscle atrophy and weak-
ness observed in muscular dystrophy type 1, the disease for
which its involvement is best known [38-40]. CELF1 is
therefore predicted to be one potential modifier of CGG
repeat-mediated neurodegeneration. Preliminary findings in
mice show modulation of neuropathological phenotypes
previously reported in the L7CGG90 transgenic mice
when the expression of CELF1 is altered (Zalewski *et al.*
Abstracts of the 1st Premutation Meeting, Perugia,
Italy, 2013). Such findings support an RNA toxicity
mechanism (see Evidence for current disease models
section), specifically that the sequestration of such
proteins within an inclusion inhibits their normal function,
leading to dysregulation (at least at the level of RNA
processing) in the cell and, over time, cell death.

Fmr1 overexpressing mice

Levels of *FMR1* mRNA bearing an expanded CGG are
elevated several fold in premutation carriers and in pa-
tients with FXTAS, supporting the hypothesis that path-
ology is the result of *FMR1* mRNA toxicity. However,
the possibility exists that toxicity could be due to either
the CGG repeat itself, elevated *FMR1* mRNA independ-
ent of the repeat expansion, or both. In a *Drosophila*
model of FXTAS, high expression levels of a CGG60
repeat causes formation of ubiquitin-positive inclusions
and neurodegeneration in the retina in a dosage- and
repeat length-dependent manner, whereas moderate
expression of the repeat allele results in little pathology.
These findings support the notion that overall abun-
dance of a CGG repeat molecule may be important for

generating a pathological phenotype [36]. To investigate
the potential deleterious effects produced by overexpres-
sion of *FMR1* mRNA with a normal CGG repeat length,
transgenic mice that overexpress *FMR1* mRNA bearing
a normal length CGG29 repeat have been generated [41].
The CGG29 transgenic mouse was obtained by pronuclear
injection of a construct containing the human *FMR1*
cDNA with 29 CGG repeats under control of a SV40/T7
promoter. This model results in a 20- to 100-fold increase
in *FMR1* mRNA in all tissues studied (for example, liver,
cerebral cortex and cerebellum). However, these animals
did not show significant differences from WT mice in
general activity or anxiety-related behaviors in open-field
tests. These results suggest it is expression of the ex-
panded CGG repeat that is primarily responsible for
pathology, and not overexpression of *Fmr1* mRNA *per se*.
Other transgenic mice overexpressing *FMR1* mRNA have
been made using a yeast artificial chromosome (YAC)
containing the full-length human *FMR1* gene. These YAC
mice show a 2- to 3-fold increase in expression of *FMR1*
mRNA and a 10- to 15-fold increase in FMRP compared
with control littermates [42,43]. When crossed with a
knock-out (KO) mouse model of FXS that lacks FMRP,
some of the pathological features of FXS were reversed.
Importantly there were no changes in overall brain
morphology at the light microscopic level due to overex-
pression of mRNA or protein. However, overexpression in
otherwise WT mice (that is, not KO mice) also resulted in
some abnormal behaviors, including decreased activity,
increased anxiety-like behavior and enhanced startle
response. Although the authors attributed these behavioral
effects to overexpression of FMRP, the high levels of *Fmr1*
mRNA could also have contributed to the behavioral
effects [43].

Yeast artificial chromosome transgenic mouse models of
the FPM

YAC transgenic mouse lines have also been generated to
study CGG repeat instability [44]. These mice were gen-
erated using a CGG92 allele isolated from an adult male
premutation carrier, a CGG repeat length that would be
expected to show expansion to the full mutation when
transmitted through the female germ line in humans.
The CGG92 region, including several hundred base pairs
of flanking sequence, was cloned into a YAC and puri-
fied YAC DNA was injected into FVB/N mouse oocytes
and then transplanted into foster mothers. A line of
offspring (line TG296) carrying a CGG90 repeat were
then identified. Although not yet well characterized,
these YAC mice show modest intergenerational CGG
repeat instability, expansion and contraction of one to
three trinucleotides across generations. There was no
influence of parental sex or age on transmission of the
repeat.
New mouse models

Continued development of new mouse models to study the FPM and FXTAS has resulted in generation of a doxycycline-inducible mouse line with a CGG99 repeat RNA under control of a doxycycline-responsive promoter (R. Hukema, Abstracts of the 1st Premutation Meeting, Perugia, Italy, 2013). Preliminary findings in this mouse show the presence of doxycycline-inducible ubiquitin-positive intranuclear inclusions in the hippocampus and cerebellum. This mouse is being used to determine critical periods for the onset of pathology as well as to help define molecular targets for development of future treatments.

Brain and cellular pathology

The description of brain pathology associated with FPM and FXTAS is limited by the availability of tissue for analysis. As a result, virtually all that is known about such pathology has come from studies of postmortem tissue from premutation carriers who developed FXTAS, and from findings in animal models. To date there have not been any published studies on brain pathology seen in FPM carriers without FXTAS, including if and when intranuclear inclusions and cell loss (for example, Purkinje neurons) may be occurring.

Intranuclear inclusions

The hallmark histopathology in FXTAS includes the presence of ubiquitin-positive inclusions in neurons and astrocytes that is widespread throughout the brain. As a further parallel between human FXTAS and the CGG KI mice, both show the presence of ubiquitin-positive intranuclear inclusions in many regions of the brain [24-26,45]. The CGG<sub>dut</sub> KI develops intranuclear inclusions in neurons in the cerebral cortex, olfactory nucleus, parafascicular thalamic nucleus, medial mammillary nucleus and colliculus inferior, cerebellum, amygdala and pontine nucleus cortex, hippocampus, hypothalamus, and in granule cells of the cerebellum (Figure 3) [24,28]. Inclusions in the dentate gyrus of the hippocampus are evident as early as 12 weeks of age [29]. The number of inclusions in glia, including astrocytes and Bergmann glia, and their distribution in the brain are more limited, and not as numerous as found in postmortem FXTAS brain tissue [14,25]. In addition, the size of the inclusions correlates significantly with the age of CGG<sub>dut</sub> KI mice, with smaller inclusions found in younger mice. Interestingly, the gradual increase in the size of the inclusions and the percentage of ubiquitin-positive neurons appear to parallel the progressive development of the neurological phenotype of FXTAS in humans [16]. Brain regions showing the presence of intranuclear inclusions correlate with the clinical features in patients with symptomatic FXTAS. Importantly, inclusions are not limited to the nervous system, and are found in both human FXTAS and in the CGG<sub>dut</sub>

KI mouse in a variety of other tissues, including pancreatic, thyroid, adrenal gland, gastrointestinal, pituitary gland, pineal gland, heart and mitral valve. Inclusions were also found in the testes, epididymis and kidney of patients with FXTAS, but not in the KI mice [46]. Therefore, FXTAS should be considered a multi-organ disease. Systematic analysis of these inclusions shows the presence of more than 20 proteins including ubiquitin, molecular chaperone Hsp40, 20S proteasome complex, DNA repair-ubiquitin-associated HR23B factor and SAM-68, DGCR8, and DROSHA [18,19,24,47-49]. The inclusions also contain *FMRI* mRNA, but surprisingly not FMRP [18]. Similar studies on the protein composition of inclusions found in CGG mouse models have not been carried out, but it is already apparent that there are some similarities between the inclusions in FXTAS and mouse models, including the presence of ubiquitin, SAM68, DGCR8 and lamin A/C, as well as several differences [18,19,24,27,47,50]. Purα has been detected in intranuclear inclusions in a *Drosophila* model of the premutation and overexpression can suppress CGG repeat-mediated neurodegeneration. However, purα has not yet been detected in inclusions in murine models and evidence for its presence in human inclusions is inconclusive [18,50]. Similarly, hnRNP-A2/B1 are found in the intranuclear inclusions in FXTAS [18], but little or none has been found in CGG KI mice [34]. Additional research on the composition of intranuclear inclusions in FXTAS and mouse models would clearly be of value.

Cell loss

An important neuropathological finding in human FXTAS is the presence of Purkinje cell degeneration [35]. This has also been observed in the CGG<sub>nih</sub> KI mouse, and in mice with an ectopic CGG90 repeat expansion whose expression is limited to cerebellar Purkinje neurons as shown in Figure 4 [26,34]. However, the generalized brain atrophy, including enlarged ventricles, that has been reported in some patients with FXTAS has not been systematically examined in any of the existing mouse models. Such studies need to be carried out using structural magnetic resonance imaging and quantitative stereology of neurons in brain regions known to be affected in FXTAS, to establish whether similar pathology also occurs in mouse models.

White matter disease

FXTAS is also characterized by white matter disease, including loss of glial cells, enlarged astrocytes, spongiosis and pallor in subcortical and cerebellar white matter, including in the middle cerebellar peduncle [14,35,51]. Additional pathology in FXTAS is seen on T2-weighted magnetic resonance images that show hyperintensities in white matter tracts, including the middle cerebellar peduncle [52]. Tractography studies using diffusion-weighted magnetic resonance imaging have provided additional
evidence for degeneration in major white matter fibers
tracts in FXTAS, including the middle cerebellar peduncle,
superior cerebellar peduncle and corpus callosum, that
was not found in premutation carriers without FXTAS
[51]. As yet, these important findings have not been
systematically examined in mouse models of the FPM or
FXTAS, and there are no published reports of white
matter pathology or degeneration of major fiber tracts in
animal models.

Dendrite and dendritic spine morphology

Studies of Golgi-stained neurons have also revealed ultra-
structural changes in dendrites and dendritic spines in
both CGG<sub>dut</sub> and CGG<sub>nih</sub> KI mice [30,53]. The CGG<sub>dut</sub> KI
mouse shows fewer dendritic branches proximal to the
soma, reduced total dendritic length and longer dendritic
spines on basilar, but not on apical dendrites in pyramidal
neurons in the primary visual cortex. Neither total den-
dritic spine density, nor the density for specific dendritic
spine subtypes (that is, stubby, mushroom, filipodial) dif-
fered between WT and KI mice. Dendrite and dendritic
spine morphology has also been examined in CGG<sub>nih</sub> KI
mice in several brain regions, including the medial pre-
frontal cortex, hippocampus and basal lateral amygdala. In
all three brain regions, the branching complexity of apical
and basilar dendrites was significantly lower and spines
were longer in KI mice compared to WT, consistent with
findings in the CGG<sub>dut</sub> KI mouse. However, in the CGG<sub>nih</sub>
KI mouse, dendritic spine density was generally increased
in all three brain regions in contrast to the CGG<sub>dut</sub> KI
mouse, which did not show changes in spine density. It is
interesting to note that longer dendritic spines found
in the cortex of CGG KI mice have also been reported in
Golgi studies of postmortem tissue in FXS [54,55] and in
*Fmr1* KO mice [56,57], whereas the reduction in dendritic
branching complexity in CGG KI mice was not found in
the *Fmr1* KO mouse [56]. The reasons for these similar-
ities and differences are unknown but should be further
investigated. To our knowledge, dendritic branching and
spine morphology have not been examined in postmortem
tissues from carriers of the FPM or patients with FXTAS.

Lamin A/C disruption

Expression of expanded CGG RNA also results in the
widespread disruption of lamin A/C proteins with asso-
ciated abnormalities in nuclear envelope morphology
*in vitro* and *in vivo* [58,59]. Lamins A/C are intermediate
filament proteins that that line the inner nuclear mem-
brane where they help maintain the shape and mechan-
ical integrity of the nucleus [60]. They are generated
from a single *LMNA* gene by alternative splicing, and
mutations have been linked to a variety of neurodegenera-
tive diseases. Cells deficient in lamin A/C show decreased
survival and defective response to DNA damage [61].

These observations suggest that FXTAS may result in
a functional laminopathy. This is consistent with recent
findings that demonstrate that laminopathy diseases, in-
cluding restrictive dermopathy and Hutchinson-Gilford
progeria syndrome, result in increased levels of reactive
oxygen species and accumulation of DNA damage [62].
Moreover, several proteins involved in telomere main-
tenance [63-65] are present in the intranuclear inclu-
sions characteristic of FXTAS (for example, lamin A/C,
Ku80, γH2AX) [18] and could account for shorter
telomere length demonstrated in patients with FXTAS
[66,67]. Shorter telomere length could also contribute to
the reduce life expectancy associated with longer CGG
repeat lengths in patients with FXTAS [14,25]. While
disruption of nuclear lamin A/C architecture has been
reported in mouse embryonic fibroblasts from CGG<sub>dut</sub>
KI mice, studies in mice examining Ku80 and γH2AX
have not been carried out [58].

Mitochondrial dysfunction

Several symptoms reported in FXTAS share some com-
monalities with mitochondrial respiratory chain enzyme
deficiencies, including gait ataxia, white matter disease,
peripheral neuropathology, muscular weakness and neuro-
psychiatric disorders [68]. Mitochondrial dysfunction
occurs in FPM and FXTAS and has been examined in
cultured skin fibroblasts and in frozen frontal cortex
from postmortem brain tissue samples from premuta-
tion carriers with or without FXTAS [68]. Decreased
NAD- and FAD-linked oxygen uptake rates have been
found in premutation carriers compared to controls. In
addition there is reduced expression of the mitochon-
drial protein MnSOD, an antioxidant enzyme, and nitra-
tion of ATPB, a putative marker for nitrative/oxidative
stress is elevated approximately 2-fold in FPM and
FXTAS compared to controls, indicating mitochondrial
dysfunction. Mitochondrial dysfunction has also been
found in cultured hippocampal neurons isolated from
CGG<sub>dut</sub> KI mice as early as 4 days *in vitro* (DIV) [69].
Density and mobility were assessed by time-lapse im-
aging of mitochondria labeled with Mitotracker Red
CMXRos, and oxygen consumption was estimated by
measuring the rate of change of dissolved O<sub>2</sub> in the
medium surrounding the cultured hippocampal neurons
using a Seahorse Bioscience extracellular flux analyzer.
CGG<sub>dut</sub> KI mice showed reduced density of mitochon-
dria in proximal neurites (that is, within 25 µm of
soma), as well as significantly reduced mobility com-
pared to WT mice. Neurons from CGG<sub>dut</sub> KI mice also
showed high basal oxygen consumption rates and evi-
dence for increased protein leakage and higher ATP
production. The authors suggested that these abnormal-
ities in mitochondrial distribution and bioenergetics
may contribute to previous reports of lower viability
and reduced dendritic branching of cultured hippocam-
pal neurons [70] as well as to reduced dendritic branch-
ing and altered spine morphology in CGG KI mouse
neocortex [30,53]. It is important to consider the possi-
bility that mitochondrial disease may contribute to the
risk for premutation carriers to become symptomatic or
to develop FXTAS, and this potential link should be
explored in futures studies in using mouse models.

Molecular findings

Fmr1 mRNA and FMRP

Both the CGG<sub>dut</sub> KI and the CGG<sub>nih</sub> KI mice have proven to be very useful models to study the molecular aspects of the expanded CGG repeat. The brains of these two mouse lines show small (10% to 30%) to moderate (>50%) reductions in FMRP, respectively, despite the fact that 2- to 3-fold elevated levels of *Fmr1* mRNA are found [23,26,28,71-73]. These results parallel to a great extent what is found in some human premutation carriers and in patients with FXTAS as outlined in Table 1 [21]. The linear correlation between *FMR1* mRNA levels and the repeat size in FPM and in patients with FXTAS [72,74] has also been found in brain tissue from the CGG<sub>dut</sub> KI mouse [73]. Entezam *et al*. were able to show a direct relationship between CGG-CCG repeat size and *Fmr1* mRNA levels in the brains of the CGG<sub>nih</sub> KI mice, although the number of mice studied for the different repeat sizes was limited [26]. The cellular mechanism underlying the increase in *Fmr1* mRNA levels is unknown, but could be due to a feedback mechanism resulting from reduced levels of FMRP. Mechanisms underlying reduced FMRP include impeded migration of the 40S ribosomal complex along the expanded CGG tract, as well as the use of an alternative internal ribosome entry site for initiation of translation. An internal ribosome entry site has been identified in the 5'UTR of *FMR1* mRNA [75].

Fmr1 splice variants and FMRP isoforms

The *FMR1* gene has 17 exons with alternative splice sites on exons 12, 14, 15 and 17 that result in the expression of multiple FMRP isoforms [76-78]. The splicing pattern of these isoforms is of interest as, in some isoforms, the truncation or absence of functional domains would suggest a change in FMRP functional properties including its selection of protein partners and mRNA targets and its cellular localization. For instance, the N-terminus of FMRP harbors a nuclear localization signal and *FMR1* mRNA binding activity is driven by two K Homology domains encoded by exons 8 to 12 and an RGG box domain in exons 14 to 15 [79]. Additionally, a nuclear export signal is localized to exon 14 and serine phosphorylation sites involved in translational regulatory activity of FMRP as well as methylation sites are also localized to exon 15.

The transcript levels of these isoforms are developmen-
tally regulated in the brain of the WT C57BL/6 mouse
strain [77], the same strain used to construct the CGG<sub>dut</sub>
KI mouse model [24]. Isoform distributions were similar
across 11 different brain regions with the exception of the
hippocampus and the olfactory bulb. Although to date no
information is available on isoform distribution in the
CGG<sub>dut</sub> KI mouse, the polyadenylation state of *Fmr1* tran-
scripts, which can be informative for the stability and the
translational efficiency of the mRNA, has been investi-
gated in these mice. The CGG<sub>dut</sub> KI mouse exhibits an
increased population of short poly(A) mRNAs, usually
indicative of inefficiently translated transcripts, compared
to WT [80]. It would be interesting to know whether
particular mRNA isoforms are thus more efficiently
translated than others in the CGG<sub>dut</sub> KI background.

Expression profiling

Dysfunction of the GABAergic system has been reported
in CGG<sub>dut</sub> KI mice [81]. Specifically, overexpression of
genes for several GABA<sub>A</sub> receptor subunits (for example,
α1,3,4; β2; γ2) and proteins involved in GABA metabolism
(gad1, ssadh) has been observed in the cerebellum, but
not the cortex, of CGG<sub>dut</sub> KI mice, which could be related
to the motor phenotype observed in FXTAS [82,83]. In
*Fmr1* KO mice, expression was decreased for some of
these same genes (for example, *gad1*, *ssadh*), but the rea-
sons for this difference are unclear. Microarray analysis in
the cerebellum of transgenic mice that overexpress human
*FMR1* with a normal range CGG29 repeat has also been
carried out, but there were no clear changes in the
GABAergic system compared to controls. Among GABA-
related genes, only up-regulation of the GABA<sub>A</sub> receptor-
associated protein-like 2 (*Gabarapl2*) gene was observed
[41]. These results provide additional support that path-
ology in CGG KI mice, at least in the GABA system, is
due to expansion of CGG repeats rather than increased
mRNA levels, given that *FMR1* mRNA levels were
increased 20 to 100 times in these transgenic mice com-
pared with those of WT littermates. However, other
changes were seen in the transcriptome of these mice
that could be a consequence of an overabundance of
*FMR1* mRNA. Interestingly, the two most altered genes
in the transcriptome were *transthyretin* (*Trt*), and *serpina3*,
putative biomarkers for Alzheimer's disease [84,85]. Ser-
pina3, a serine protease inhibitor that is released during
inflammatory responses, was up-regulated and may reflect
the increased prevalence of autoimmune disease (for
example, lupus, multiple sclerosis, fibromyalgia, thyroid
disease) in females with the *FMR1* premutation [86].
Transthyretin, a transport protein for retinol and thyroxine
thought to contribute to thyroid hormone homeostasis,
was down-regulated [87]. Although speculative, reduced
transcription could be related to the hypothyroidism
reported in some patients with FXTAS [3]. In addition, two microRNAs, mir-181a-1 and let-7 appeared up-regulated in CGG mice. Up-regulation of Let-7 miRNA has been also been reported in a *Drosophila* model of FXTAS [88]. This is important because several miRNAs are up-regulated in human premutation carriers [89], although they differed from those observed in CGG transgenic mice [41].

Electrophysiological findings

GABA/glutamate imbalance and abnormal synaptic network activity

The origin of pathology in FXS and in some FPM car-
riers, with or without FXTAS mutations, is the presence
of a CGG repeat expansion on *FMR1*, raising the possi-
bility that some of the same molecular pathways could
be affected in both disorders, and those associated with
glutamatergic signaling in particular [1,74,90,91]. This is
in spite of differences in the causal molecular underpin-
nings in the disorders, and specifically the lack of FMRP
expression in FXS versus the overexpression of *FMR1*
mRNA in the FPM and FXTAS. In fact, the dysregula-
tion in excitatory and inhibitory neurotransmission in
the central nervous system of FXS KO mice has been
the subject of active investigation during the last decade,
and evidence has recently surfaced that suggests a
similar dysregulation in the CGG KI mice [1,90,91].

Hippocampal CGG<sub>dut</sub> KI neurons *in vitro* show a devel-
opmental defect in connectivity and impaired dendritic
growth observed at 7 and 21 days DIV. There is also a loss
of cell viability, also suggestive of a neurodegenerative
component to the FPM [70]. Interestingly, in the same
neurons, the expression of the vesicular GABA and glu-
tamate transporters VGAT and VGLUR1, respectively, is
reduced at 21 DIV, but not at 7 DIV. These alterations are
associated with a 4- to 8-fold increase in *Fmr1* mRNA and
an approximately 50% decrease in FMRP.

Abnormal patterns of electrical activity are also seen
*in vitro* in hippocampal neurons from CGG<sub>dut</sub> KI mice, in-
cluding enhanced clustered burst (CB) firing. Specifically,
hippocampal neurons cultured from CGG<sub>dut</sub> KI mice
display CB electrical spiking activity and abnormal patterns
of spontaneous synchronous Ca<sup>2+</sup> oscillations under basal
culture conditions [92]. The principal mechanisms contrib-
uting to these neuronal network defects in basal electrical
activity appear to be associated with a gain of function in
type I metabotropic glutamate receptors (mGluRs) and/or
a loss of function in GABA<sub>A</sub> receptor signaling. This con-
clusion is supported by data indicating that: the type I mGluR
receptor agonist 3,5-Dihydroxyphenylglycine (DHPG), but
neither NMDA nor AMPA receptor agonists, increased
CB firing patterns in WT neurons with increased spike
rate and mean burst duration similar to those observed in
FPM hippocampal neurons; selective mGluR1/5 antagonists

7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) and 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP) abrogated abnormal electrical activity in FPM neurons; FPM astrocytes have impaired glutamate uptake [69,93]; WT cultures exposed to the astrocyte glutamate transport competitive antagonist DL-threo-β-benzyloxyaspartic acid produced electrical firing patterns indistinguishable from those of CGG<sub>dut</sub> KI neurons; GABA<sub>A</sub> receptor block with microtoxin generated CB firing behavior observed in CGG<sub>dut</sub> neurons; and the allosteric GABA<sub>A</sub> receptor enhancer allopregnanolone essentially restored WT electrical spiking patterns.

These functional deficits are directly pertinent to the
altered patterns of neuronal complexity reported earlier
using the same *in vitro* CGG<sub>dut</sub> KI model [70]. Neuronal
network activity is essential for normal neuronal migra-
tion, dendritic growth and synaptic plasticity, processes
mediated by spatially and temporally orchestrated intra-
cellular Ca<sup>2+</sup> signals. Therefore, the abnormal CB elec-
trical activity and abnormal patterns of spontaneous Ca<sup>2+</sup>
oscillations observed in hippocampal neurons from
CGG<sub>dut</sub> KI mice are likely to contribute, at least in part, to
impaired dendritic growth and synaptic architecture.

Hippocampal synaptic plasticity

Deficits in processing spatial and temporal information
have been reported in FPM carriers and in patients with
FXTAS, suggesting hippocampal-associated pathology.
In order to fully characterize the CGG KI mouse and to
provide clues to which brain regions mediate these
cognitive deficits (for example, hippocampus), *in vitro*
studies of synaptic plasticity in acute hippocampal slices
isolated from CGG<sub>dut</sub> KI mice and WT mice have been
carried out. Specifically long-term potentiation (LTP) of
synaptic transmission and long-term synaptic depression
(LTD) in CGG<sub>dut</sub> and WT mice have been examined.
The results demonstrated that the magnitude of LTP
was significantly lower in CGG KI mice compared to
WT mice, indicating impaired synaptic plasticity. Simi-
larly, LTD, whether induced by low-frequency electrical
stimulation (1 Hz) or bath application of the mGluR1/5
agonist DHPG, was also limited in CGG KI mice versus
WT mice. These findings implicate loss of neuroplasti-
city in the hippocampus in the spatial and temporal cog-
nitive deficits associated with CGG repeat expansions
and the neurological pathology in FXTAS [94]. By con-
trast, enhanced LTD has been reported in the CGG<sub>nih</sub>
KI mouse model [95]. LTD at CA3-CA1 hippocampal
synapses induced by bath application of the group I
mGluR agonist DHPG was enhanced relative to that
seen in WT littermates. *Fmr1* mRNA production was
increased, FMRP translational efficiency in response to
DHPG was impaired, and basal FMRP levels were
moderately reduced. The authors noted that *Fmr1* KO mice completely lacking FMRP also showed enhanced LTD, suggesting that the enhanced LTD in the CGG*nih* KI mouse may be due, at least in part, to lower levels of FMRP. The differing results for LTD between the CGG*dut* and CGG*nih* KI mouse models may therefore be the result of small versus moderate reductions in FMRP, respectively, indicating different cellular mechanisms for the differing results.

## Developmental aspects in FPM and FXTAS

FXTAS was originally described as a late-onset neurodegenerative disorder typically appearing in premutation carriers in the fifth or sixth decade of life. However, it is clear from both human [96,97] and mouse studies [29] that the consequences of the expanded CGG repeat can be seen in FPM carriers much earlier in development, indicating that the disease process likely begins much earlier in life, and possibly as early as during gestation [98]. Some children with the premutation have been reported to show cognitive deficits and behavioral problems, including symptoms of autism spectrum disorder and attention-deficit hyperactivity disorder [96,97]. Young (<12-week-old) CGG*dut* and CGG*nih* KI mice show impaired processing of spatial information [29] and abnormal locomotor activity and anxiety in the elevated Plus-maze [30].

The possibility that the premutation may affect early brain development is supported by findings in the CGG*dut* KI mouse, where abnormal migration and differentiation of neuronal precursors during development of the embryonic cortical plate has been found [98]. In this study, precursor cells and embryonic neurons were labeled *in utero* on embryonic day 14 (E14) by intracerebral injections of a retrovirus encoding EGFP. The entire cell body, cytoplasm and processes of infected cells and their progeny were labeled with the EGFP reporter. The morphology of EGFP-labeled radial glial cells and immature neurons was not different between KI and WT neurons when examined on E17. However, there was evidence for altered differentiation of embryonic neural progenitor cells in the developing neocortex.

Radial glial cells in the ventricular zone express the transcription factor Pax6, divide at the ventricular surface and give rise to intermediate neuronal progenitor cells that express the transcription factor Tbr2 [99,100]. The CGG*dut* KI mice had a greater number of Pax6⁺ cells in the ventricular zone and fewer Tbr2⁺ cells in the subventricular zone than WT mice, suggesting that delayed differentiation of the Pax6 cells in the CGG*dut* KI mice may have produced a shift towards more Pax6⁺ and fewer Tbr2⁺ cells. Importantly, the shift in cell distribution could not be attributed to increased proliferation of Pax6⁺ cells, decreased proliferation of Tbr2⁺ or increased cell death among Tbr2⁺ cells. These data suggest that the *Fmr1* CGG repeat allele impacts the developing brain during

gestation, much earlier than previously realized, and point to a neurodevelopmental component in FXTAS.

## Neurobehavioral correlates

Key features of patients with FXTAS are late-onset ataxia and memory impairments. Similar phenotypes have been found for CGG KI mice. Motor performance on the rotarod declines with age in CGG*dut* KI mice [31]. In addition, sensory-motor coordination is impaired in adult CGG*dut* KI animals when they are required to traverse a horizontal ladder (the ladder rung task). Both male and female CGG*dut* KI mice showed impairments that were positively correlated with CGG repeat size [101]. Poor performance in the rotarod and ladder rung test may reflect the ataxia seen in FXTAS. Adult female CGG KI mice are also impaired in learning a skilled forelimb motor task, in which they are trained to reach through a narrow opening in a Plexiglas box to grasp and obtain a small food reward positioned just outside. Again, performance was worse with longer CGG repeat lengths [102]. Similar experiments have not yet been carried out in male mice. To date, studies in CGG KI mice have not reported intention tremors, a key neurological feature in FXTAS. The reason for this is unclear, but may be related to the quadrupedal organization of the rodent motor system.

Spatial learning and memory in the Morris water maze is impaired in CGG*dut* KI mice at 52 weeks of age, but not at 20 weeks, indicating a progressive nature of the deficit [31]. Additional spatial deficits in CGG*dut* KI mice are seen in the 'metric' spatial processing test, which involves processing precise angles and distances that separate objects in space, without regard to the identity of the objects [103]. In this test, mice are allowed to explore two identical objects separated in space by a fixed distance for 15 min (the study phase), showing very little further exploration at the end of this time. Mice are removed from the apparatus, the distance between the objects changed (for example, moved closer together), and the mice are allowed to re-explore the objects for 5 min (the test phase). During the test phase, WT mice showed increased object exploration, indicating that they detected a change in the distance between objects, whereas CGG*dut* KI mice failed to re-explore the objects. Deficits in this task were seen as early as 12 weeks of age, when small but easily detectable intranuclear inclusions were present in neurons in the dentate gyrus of the hippocampus but not in the parietal cortex [29]. Lesion studies have implicated the dentate gyrus and CA3 hippocampus in processing of metric spatial information, a form of spatial pattern separation [104]. This suggests that histopathology (for example, presence of intranuclear inclusions, altered dendritic and spine morphology) in the dentate gyrus and CA3 subregion of the hippocampus in
CGG KI mice may contribute to this spatial processing
deficit. Although the role of intranuclear inclusions to
pathology in FXTAS is unclear, the presence of intra-
nuclear inclusions in different brain regions at different
ages appears to follow a similar time course as the
emergence of behavioral dysfunction in the CGG KI
mouse, suggesting there might be a relationship
between spatial deficits and inclusion formation
[24,105,106].

Additional behavioral pathology found in the CGG<sub>nih</sub>
KI mouse model of the FPM and FXTAS includes mild
hyperactivity, decreased anxiety in elevated plus maze,
and impaired shock avoidance learning [30].

Evidence for current disease models

RNA toxicity

Studies in mouse models have been particularly useful in
identifying molecular mechanisms in the FPM and
FXTAS. An RNA 'toxic gain of function' mechanism has
been proposed in which elevated *FMRI* mRNA tran-
scripts bearing an expanded CGG repeat are cytotoxic.
Toxicity appears to be the result of the expanded CGG
repeat *per se*, and not of overexpression of *FMRI*.
This is supported by the fact that ectopic expression of a
CGG repeat expansion in the premutation range is suffi-
cient to induce formation of intranuclear inclusions, reduce
cell viability, trigger neuronal death (for example, Purkinje
cell loss) and produce behavioral deficits [34,59,107],
whereas overexpression of *Fmr1* mRNA without a CGG
repeat expansion does not appear to be toxic [41]. Similar
RNA toxicity has been suggested to underlie the path-
ology in several repeat diseases, including the myotonic
muscular dystrophies. In this model, sequestration of
important proteins through their interactions with ex-
panded repeats prevents the proteins from carrying out
their normal functions. As shown in Figure 2A, a similar
protein sequestration mechanism has been proposed to
underlie disease processes in the FPM and in FXTAS
[2,36,82,108]. Based on studies in human and animal
(for example, mouse, fly) tissues, a number of candidate
RNA binding proteins have been identified, including
DGCR8 and DROSHA [47], SAM68 [19], purα [109,110],
hnRNPA2/B1 and CUGBP1 [37].

Sequestration of DROSHA/DGCR8 and miRNAs

While the evidence is strong for the binding of proteins
to CGG expansions and sequestration of proteins within
ubiquitin-positive inclusions, the consequences of seques-
tration for cell function remain to be described. However,
a recent study has linked sequestration of proteins asso-
ciated with miRNA processing with the disease process
in FXTAS [47]. Specifically, the double-stranded RNA-
binding protein DGCR8 binds preferentially to CGG repeats
of pathogenic length (that is, CGG repeat length >60). As

depicted in Figure 2A, this leads to partial sequestration of
DGCR8 and its binding partner DROSHA to expanded
CGG repeats within CGG RNA aggregates. DGCR8 and
DROSHA are important for processing pre-miRNAs into
mature miRNAs by the DICER enzyme. Dgcr8 deficiency
in heterozygous Dgcr8+/− mice results in reduced syn-
aptic potentiation in layer five pyramidal neurons in the
medial prefrontal cortex of mice [111]. Large deletions
in the 22q11 locus, which include Dgcr8, result in
altered dendritic spine morphology, reduced dendritic
branching complexity and impaired working memory
[112]. Similarly, loss of DICER in mice results in pro-
gressive neuronal degeneration [113], reduced dendritic
branching and increased dendritic spine length [114],
ataxia, and reduced brain size following deletion from
striatal neurons [115]. These results suggested a model
in which double-stranded CGG RNA forms hairpins
[91] that mimic the RNA structure of pre-miRNAs recog-
nized by DGCR8 [47]. DGCR8 and its partner DROSHA
bind to the expanded CGG repeat element and are there-
fore sequestered, reducing the production of mature miR-
NAs causing neuronal dysfunction and death [47]. This
possibility is supported by the observation that the expres-
sion of mature miRNAs was decreased in postmortem
brain samples from patients with FXTAS. In addition,
in vitro overexpression of DGCR8 restored normal den-
dritic growth and branching, and alleviated cell death of
cultured neurons expressing a toxic 60 CGG repeat [47].

Repeat associated non-AUG translation

An additional mechanism of toxicity is shown in Figure 2B.
In this model, toxicity is triggered by CGG RAN transla-
tion [20]. This is based on evidence that trinucleotide
repeats can be translated into protein even if they do not
reside in an AUG-initiated open reading frame [116], and
such translation can occur in all three possible open read-
ing frames of a transcript generating multiple potentially
toxic products from a single repeat [117]. In the case of
FXTAS, it has been proposed that RAN translation initi-
ated in the 5'UTR of *FMRI* mRNA results in the produc-
tion of a cytotoxic polyglycine-containing protein named
FMRpolyG [20]. This is supported by results from human
FXTAS and animal model studies. Specifically, the pres-
ence of the FMRpolyG was confirmed by western blot in
cerebellar lysates of postmortem FXTAS brains. FMRpo-
lyG staining was specific for FXTAS, and was not found in
control brains, or in brain sections from patients with spi-
nocerebellar ataxia type 3 or Alzheimer's disease. Interest-
ingly, there were clear differences between the CGG<sub>dut</sub> KI
and CGG<sub>nih</sub> KI mouse models, with co-localization of
FMRpolyG and ubiquitin-positive intranuclear inclusions
in the cortex and hypothalamus of the CGG<sub>dut</sub> KI mouse,
but not in the CGG<sub>nih</sub> KI mouse. These data suggest that
some of the differing pathology between the two mouse
models could be explained by differences in the ability to generate the toxic polyglycine peptide. The mechanisms underlying RAN translation are as yet unknown, but the presence of the polyglycine peptide (that is, FMRpolyG) in FXTAS and the CGG KI mouse models led to the proposal by Todd et al. that a scanning 43S ribosomal pre-initiation complex stalls at the CGG repeat, resulting in use of an alternative non-AUG start site for translation in the +1 reading frame (that is, GGC, polyglycine) and the production of the FMRpolyG protein. The data did not show translation product from the +0 (that is, CGG, polyarginine) reading frame, but some, albeit less efficient, translation in the +2 (that is, GCG, polyalanine) reading frame was observed [20].

## Conclusions

Although uniquely human components of disease cannot be fully captured in other species, mouse models of FXTAS have provided useful research tools to test hypotheses about the causes of the disorder, and to discover effective treatments. Development of CGG KI mice has provided insight into the natural history of the disorder, the molecular correlates, hallmark pathology in the brain and other organ systems, as well as an understanding of the neurobehavioral effects of expression of CGG repeat expansions. These KI mice now allow for the evaluation of novel therapeutic strategies, whether pharmacological or gene-targeted, to halt or reverse disease processes and to improve neurological outcome. Ongoing development of new mouse lines, including conditional and inducible mice, should further increase the value of animal models to understand the pathology of repeat disorders such as FXTAS. There are many open questions to be answered that will continue to rely on mouse models, including why mRNA levels are elevated, the importance of reduced FMRP in pathology, whether intranuclear inclusions are toxic or simply mark the progress of disease, and how protein sequestration and RAN translation contribute to the disease process in FPM and FXTAS.

## Abbreviations

AMPA: α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; ATPB: β-subunit of ATP synthase; CB: clustered burst; DHPG: 3,5-dihydroxyphenylglycine; DIV: days *in vitro*; EGFP: enhanced green fluorescent protein; FAD: flavin adenine dinucleotide; FMRP: fragile X mental retardation protein; FPM: fragile X premutation; FXS: fragile X syndrome; FXTAS: fragile X-associated tremor/ataxia syndrome; KI: knock-in; KO: knock-out; LTD: long-term synaptic depression; mGluR: metabotropic glutamate receptors; miRNA: microRNA; MnSOD: manganese superoxide dismutase; NMDA: N-methyl-d-aspartate; NAD: nicotinamide adenine dinucleotide; UTR: untranslated region; WT: wild-type; YAC: yeast artificial chromosome.

## Competing interests

DLN patented a method for detecting FMR1 mutation: US PTO 6,107,015 diagnosis of the fragile X syndrome. All other authors declare that they have no competing interests.

## Authors' contributions

All of the authors contributed to the conceptualization and the writing of this review. All authors read and approved the final manuscript.

## Acknowledgements

The assistance of Andreea Pop with the editing of this manuscript is appreciated. The following funding sources supported the writing of this manuscript: NS079775 (RFB, RW); NICHD P30 HD24064-15 S1 (DLN); Dutch Brain Foundation (RW); the Intramural Program of NIDDK, NIH (KU); NIEHS ES011269 and DOD PR120921(INP); E-Rare grant (RKH); Belgian National Fund for Scientific Research, Flanders (RFK), Ministry of Health, Spain (EP).

## Author details

¹Department of Neurological Surgery, Room 502C, UC Davis, 1515 Newton Court, Davis, CA 95618, USA. ²Department Clinical Genetics, Erasmus MC, Rotterdam, Netherlands. ³NIDDK, National Institutes of Health, Bethesda, MD, USA. ⁴University of Seville, School of Medicine, Seville, Spain. ⁵Department of Medical Genetics, University of Antwerp, Antwerp, Belgium. ⁶UC Davis M.I.N. D. Institute, Sacramento, CA, USA. ⁷Department Molecular Biosciences, UC Davis, Davis, CA, USA. ⁸Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA. ⁹Department of Translational Medicine, IGBMC, Illkirch, France.

Received: 22 October 2013 Accepted: 29 January 2014

Published: 30 July 2014

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doi:10.1186/1866-1955-6-25

Cite this article as: Berman *et al.*: Mouse models of the fragile X premutation and fragile X-associated tremor/ataxia syndrome. *Journal of Neurodevelopmental Disorders* 2014 6:25.

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