Ronald A.M. Buijsen

AFQ056, a new mGluR5 antagonist for treatment of fragile X syndrome.

Fragile X syndrome, the most common form of inherited intellectual disability, is caused by a lack of FMRP, which is the product of the Fmr1 gene. FMRP is an RNA-binding protein and a component of RNA-granules found in the dendrites of neurons. At the synapse, FMRP is involved in regulation of translation of specific target mRNAs upon stimulation of mGluR5 receptors. In this study, we test the effects of a new mGluR5 antagonist (AFQ056) on the prepulse inhibition of startle response in mice. We show that Fmr1 KO mice have a deficit in inhibition of the startle response after a prepulse and that AFQ056 can rescue this phenotype. We also studied the effect of AFQ056 on cultured Fmr1 KO hippocampal neurons; untreated neurons showed elongated spines and treatment resulted in shortened spines. These results suggest that AFQ056 might be a potent mGluR5 antagonist to rescue various aspects of the fragile X phenotype.

Chronic administration of AFQ056/Mavoglurant restores social behaviour in Fmr1 knockout mice

Fragile X syndrome is caused by lack of FMR1 protein (FMRP) leading to severe symptoms, including intellectual disability, hyperactivity and autistic-like behaviour. FMRP is an RNA binding protein involved in the regulation of translation of specific target mRNAs upon stimulation of metabotropic glutamate receptor 5 (mGluR5) at the synapse. The absence of FMRP leads to enhanced activity of mGluR5 signal transduction pathways. Many conflicting results have been reported regarding social behaviour deficits in Fmr1 knockout mice, and little is known about the involvement of mGluR5 pathways on social behaviour. In this study, a three-chambered task was used to determine sociability and preference for social novelty in Fmr1 knockout mice. Disruption of Fmr1 functioning resulted in enhanced interaction with stranger mouse during sociability while no significant changes were observed during preference for social novelty assay. Chronic administration of a specific mGluR5 antagonist, AFQ056/Mavoglurant, was able to restore sociability behaviour of Fmr1 knockout mice to levels of wild type littermates. These results support the importance of mGluR5 signalling pathways on social interaction behaviour and that AFQ056/Mavoglurant might be useful as potential therapeutic intervention to rescue various behavioural aspects of the fragile X phenotype.

Rescue of dendritic spine phenotype in Fmr1 KO mice with the mGluR5 antagonist AFQ056/Mavoglurant

Fragile X syndrome (FXS) is the leading monogenic cause of intellectual disability and autism. The disease is a result of lack of expression of the fragile X mental retardation protein. Brain tissues of patients with FXS and mice with FMRP deficiency have shown an abnormal dendritic spine phenotype. We investigated the dendritic spine length and density of hippocampal CA1 pyramidal neurons in 2-, 10-, and 25-week-old Fmr1 knockout (KO). Next, we studied the effects of long-term treatment with an mGluR5 antagonist, AFQ056/Mavoglurant, on the spine phenotype in adult Fmr1 KO mice. We observed alterations in the spine phenotype during development, with a decreased spine length in 2-week-old Fmr1 KO mice compared with age-match wild-type littermates, but with increased spine length in Fmr1 KO mice compared with 10- and 25-week-old wild-type controls. No difference was found in spine density at any age. We report a rescue of the abnormal spine length in adult Fmr1 KO mice after a long-term treatment with AFQ056/Mavoglurant. This finding suggests that long-term treatment at later stage is sufficient to reverse the structural spine abnormalities and represents a starting point for future studies aimed at improving treatments for FXS.

The effect of an mGluR5 inhibitor on procedural memory and avoidance discrimination impairments in Fmr1 KO mice

Fragile X syndrome (FXS) is the most common inherited form of intellectual disability. Patients with FXS do not only suffer from cognitive problems, but also from abnormalities/deficits in procedural memory formation. It has been proposed that a lack of fragile X mental retardation protein (FMRP) leads to altered long‐term plasticity by deregulation of various translational processes at the synapses, and that part of these impairments might be rescued by the inhibition of type I metabotropic glutamate receptors (mGluRs). We recently developed the Erasmus Ladder, which allows us to test, without any invasive approaches, simultaneously, both procedural memory formation and avoidance behavior during unperturbed and perturbed locomotion in mice. Here, we investigated the impact of a potent and selective mGluR5 inhibitor (Fenobam) on the behavior of Fmr1 KO mice during the Erasmus Ladder task. Fmr1 KO mice showed deficits in associative motor learning as well as avoidance behavior, both of which were rescued by intraperitoneal administration of Fenobam. While the Fmr1 KO mice did benefit from the treatment, control littermates suffered from a significant negative side effect in that their motor learning skills, but not their avoidance behavior, were significantly affected. On the basis of these studies in the FXS animal model, it may be worthwhile to investigate the effects of mGluR inhibitors on both the cognitive functions and procedural skills in FXS patients. However, the use of mGluR inhibitors appears to be strongly contraindicated in healthy controls or non‐FXS patients with intellectual disability.

Subregion-specific dendritic spine abnormalities in the hippocampus of Fmr1 KO mice

Fragile X syndrome (FXS) is the most common inherited form of mental retardation and is caused by the lack of fragile X mental retardation protein (FMRP). In the brain, spine abnormalities have been reported in both patients with FXS and Fmr1 knockout mice. This altered spine morphology has been linked to disturbed synaptic transmission related to altered signaling in the excitatory metabotropic glutamate receptor 5 (mGluR5) pathway. We investigated hippocampal protrusion morphology in adult Fmr1 knockout mice. Our results show a hippocampal CA1-specific altered protrusion phenotype, which was absent in the CA3 region of the hippocampus. This suggests a subregion-specific function of FMRP in synaptic plasticity in the brain.

Translation of Expanded CGG Repeats into FMRpolyG Is Pathogenic and May Contribute to Fragile X Tremor Ataxia Syndrome

Summary Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder caused by a limited expansion of CGG repeats in the 5′ UTR of FMR1. Two mechanisms are proposed to cause FXTAS: RNA gain-of-function, where CGG RNA sequesters specific proteins, and translation of CGG repeats into a polyglycine-containing protein, FMRpolyG. Here we developed transgenic mice expressing CGG repeat RNA with or without FMRpolyG. Expression of FMRpolyG is pathogenic, while the sole expression of CGG RNA is not. FMRpolyG interacts with the nuclear lamina protein LAP2β and disorganizes the nuclear lamina architecture in neurons differentiated from FXTAS iPS cells. Finally, expression of LAP2β rescues neuronal death induced by FMRpolyG. Overall, these results suggest that translation of expanded CGG repeats into FMRpolyG alters nuclear lamina architecture and drives pathogenesis in FXTAS.

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

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.

Induced expression of expanded CGG RNA causes mitochondrial dysfunction in vivo

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder affecting carriers of premutation forms of the FMR1 gene, resulting in a progressive development of tremor, ataxia and neuropsychological problems. The disease is caused by an expanded CGG repeat in the FMR1 gene, leading to an RNA gain-of-function toxicity mechanism. In order to study the pathogenesis of FXTAS, new inducible transgenic mouse models have been developed that expresses either 11CGGs or 90CGGs at the RNA level under control of a Tet-On promoter. When bred to an hnRNP-rtTA driver line, doxycycline (dox) induced expression of the transgene could be found in almost all tissues. Dox exposure resulted in loss of weight and death within 5 d for the 90CGG RNA expressing mice. Immunohistochemical examination of tissues of these mice revealed steatosis and apoptosis in the liver. Decreased expression of GPX1 and increased expression of cytochrome C is found. These effects were not seen in mice expressing a normal sized 11CGG repeat. In conclusion, we were able to show in vivo that expression of an expanded CGG-repeat rather than overexpression of a normal CGG-repeat causes pathology. In addition, we have shown that expanded CGG RNA expression can cause mitochondrial dysfunction by regulating expression levels of several markers. Although FTXAS patients do not display liver abnormalities, our findings contribute to understanding of the molecular mechanisms underlying toxicity of CGG repeat RNA expression in an animal model. In addition, the dox inducible mouse lines offer new opportunities to study therapeutic interventions for FXTAS.

Ultrastructural analysis of the functional domains in FMRP using primary hippocampal mouse neurons

Fragile X syndrome is caused by lack of the protein FMRP. FMRP mediates mRNA binding, dendritic mRNA transport and translational control at spines. We examined the role of functional domains of FMRP in neuronal RNA-granule formation and dendritic transport using different FMRP variants, including the mutant FMRP_I304N and the splice-variant FMRP_Iso12. Both variants are absent from dendritic RNA-granules in Fmr1 knockout neurons. Co-transfection experiments showed that wild-type FMRP recruits both FMRP variants into dendritic RNA-granules. Co-transfection of FXR2, an FMRP homologue, also resulted in redistribution of both variants into dendritic RNA-granules. Furthermore, the capacity of the variants to transport their mRNAs and the mRNA localization of an FMR1 construct containing silent point-mutations affecting only the G-quartet-structure were investigated. In conclusion, we show that wild-type FMRP and FXR2P are able to recruit FMRP variants into RNA-granules and that the G-quartet-structure in FMR1 mRNA is not essential for its incorporation in RNA-granules.

Presence of inclusions positive for polyglycine containing protein, FMRpolyG, indicates that repeat-associated non-AUG translation plays a role in fragile X-associated primary ovarian insufficiency.

STUDY QUESTION Does repeat-associated non-AUG (RAN) translation play a role in fragile X-associated primary ovarian insufficiency (FXPOI), leading to the presence of polyglycine containing protein (FMRpolyG)-positive inclusions in ovarian tissue? SUMMARY ANSWER Ovaries of a woman with FXPOI and of an Fmr1 premutation (PM) mouse model (exCGG-KI) contain intranuclear inclusions that stain positive for both FMRpolyG and ubiquitin. WHAT IS KNOWN ALREADY Women who carry the FMR1 PM are at 20-fold increased risk to develop primary ovarian insufficiency (FXPOI). A toxic RNA gain-of-function has been suggested as the underlying mechanism since the PM results in increased levels of mRNA containing an expanded repeat, but reduced protein levels of fragile X mental retardation protein (FMRP). Recently, RAN translation has been shown to occur from FMR1 mRNA that contains PM repeat expansions, leading to FMRpolyG inclusions in brain and non-CNS tissues of fragile X-associated tremor/ataxia syndrome (FXTAS) patients. STUDY DESIGN, SIZE, DURATION Ovaries of a woman with FXPOI and women without PM (controls), and ovaries from wild-type and exCGG-KI mice were analyzed by immunohistochemistry for the presence of inclusions that stained for ubiquitin and FMRpolyG . The ovaries from wild-type and exCGG-KI mice were further characterized for the number of follicles, Fmr1 mRNA levels and FMRP protein expression. The presence of inclusions was also analyzed in pituitaries of a man with FXTAS and the exCGG-KI mice. PARTICIPANTS/MATERIALS, SETTING, METHODS Human ovaries from a woman with FXPOI and two control subjects and pituitaries from a man with FXTAS and a control subjects were fixed in 4% formalin. Ovaries and pituitaries of wild-type and exCGG mice were fixed in Bouins fluid or 4% paraformaldehyde. Immunohistochemistry was performed on the human and mouse samples using FMRpolyG, ubiquitin and Fmrp antibodies. Fmr1 mRNA and protein expression were determined in mouse ovaries by quantitative RT-PCR and Western blot analysis. Follicle numbers in mouse ovaries were determined in serial sections by microscopy. MAIN RESULTS AND THE ROLE OF CHANCE FMRpolyG-positive inclusions were present in ovarian stromal cells of a woman with FXPOI but not in the ovaries of control subjects. The FMRpolyG-positive inclusions colocalized with ubiquitin-positive inclusions. Similar inclusions were also observed in the pituitary of a man with FXTAS but not in control subjects. Similarly, ovaries of 40-week-old exCGG-KI mice, but not wild-type mice, contained numerous inclusions in the stromal cells that stained for both FMRpolyG- and ubiquitin, while the ovaries of 20-week-old exCGG-KI contained fewer inclusions. At 40 weeks ovarian Fmr1 mRNA expression was increased by 5-fold in exCGG-KI mice compared with wild-type mice, while Fmrp expression was reduced by 2-fold. With respect to ovarian function in exCGG-KI mice: (i) although the number of healthy growing follicles did not differ between wild-type and exCGG-KI mice, the number of atretic large antral follicles was increased by nearly 9-fold in 40-week old exCGG-KI mice (P < 0.001); (ii) at 40 weeks of age only 50% of exCGG-KI mice had recent ovulations compared with 89% in wild-type mice (P = 0.07) and (iii) those exCGG-KI mice with recent ovulations tended to have a reduced number of fresh corpora lutea (4.8 ± 1.74 versus 8.50 ± 0.98, exCGG-KI versus wild-type mice, respectively, P = 0.07). LIMITATIONS, REASONS FOR CAUTION Although FMRpolyG-positive inclusions were detected in ovaries of both a woman with FXPOI and a mouse model of the FMR1 PM, we only analyzed one ovary from a FXPOI subject. Caution is needed to extrapolate these results to all women with the FMR1 PM. Furthermore, the functional consequence of FMRpolyG-positive inclusions in the ovaries for reproduction remains to be determined. WIDER IMPLICATIONS OF THE FINDINGS Our results suggest that a dysfunctional hypothalamic-pituitary-gonadal-axis may contribute to FXPOI in FMR1 PM carriers. STUDY FUNDING/COMPETING INTERESTS This study was supported by grants from NFXF, ZonMW, the Netherlands Brain Foundation and NIH. The authors have no conflict of interest to declare.