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. 2015 Sep 1;24(17):4948-57.
doi: 10.1093/hmg/ddv216. Epub 2015 Jun 9.

Reversibility of Neuropathology and Motor Deficits in an Inducible Mouse Model for FXTAS

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Free PMC article

Reversibility of Neuropathology and Motor Deficits in an Inducible Mouse Model for FXTAS

Renate K Hukema et al. Hum Mol Genet. .
Free PMC article

Abstract

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder affecting carriers of the fragile X-premutation, who have an expanded CGG repeat in the 5'-UTR of the FMR1 gene. FXTAS is characterized by progressive development of intention tremor, ataxia, parkinsonism and neuropsychological problems. The disease is thought to be caused by a toxic RNA gain-of-function mechanism, and the major hallmark of the disease is ubiquitin-positive intranuclear inclusions in neurons and astrocytes. We have developed a new transgenic mouse model in which we can induce expression of an expanded repeat in the brain upon doxycycline (dox) exposure (i.e. Tet-On mice). This Tet-On model makes use of the PrP-rtTA driver and allows us to study disease progression and possibilities of reversibility. In these mice, 8 weeks of dox exposure was sufficient to induce the formation of ubiquitin-positive intranuclear inclusions, which also stain positive for the RAN translation product FMRpolyG. Formation of these inclusions is reversible after stopping expression of the expanded CGG RNA at an early developmental stage. Furthermore, we observed a deficit in the compensatory eye movements of mice with inclusions, a functional phenotype that could be reduced by stopping expression of the expanded CGG RNA early in the disease development. Taken together, this study shows, for the first time, the potential of disease reversibility and suggests that early intervention might be beneficial for FXTAS patients.

Figures

Figure 1.
Figure 1.
Dox-induced expression of nCGG-eGFP in the brain. (A) The Tet-On system was used to generate bigenic mice expressing an 11CGG or 90CGG repeat at the RNA level. Expression of rtTA is controlled by the PrP promoter on a separate transgene. Upon dox administration, rtTA will be activated and can bind the Tet response element on another transgene, which induces expression of the nCGG repeat at the RNA level and eGFP at the protein level. As the transgene contains the 5′-UTR of the FMR1 gene, a polypeptide is formed from the repeat by RAN translation. (B) Quantitative RT-PCR on RNA isolated from the brain of dox-treated (16–28 weeks) bigenic TRE-nCGG-eGFP/PrP-rtTA mice. 90CGG RNA levels are lower than 11CGG RNA levels in whole brain homogenates (N = 8). Error bars represent SEM.
Figure 2.
Figure 2.
eGFP expression in brain after dox induction of bigenic TRE-nCGG-eGFP/PrP-rtTA mice. Immunohistochemical staining of paraffin section of brain from dox-induced nCGG-eGFP-expressing mice in (A) hippocampus (10×), (B) mainly in the CA3 area (40×); (C) cerebellum (40×), (D) mainly in lobule X (100×) and (E) striatum 10× and (F) striatum 40×.
Figure 3.
Figure 3.
Ubiquitin and FMRpolyG-positive intranuclear inclusions in different brain areas after dox induction of bigenic TRE-90CGG-egFP/PrP-rtTA mice. (A) Ubiquitin-positive intranuclear inclusions in CA3 of the hippocampus. (B) FMRpolyG-positive intranuclear inclusions in CA3 of the hippocampus. (C) Ubiquitin-positive intranuclear inclusions in striatum. (D) FMRpolyG-positive intranuclear inclusions in striatum. (E) Ubiquitin-positive intranuclear inclusions in the granular cell layer of lobule X after 8 weeks of dox induction. (F) FMRpolyG-positive intranuclear inclusions in the granular cell layer of lobule X after 8 weeks of dox induction. (G) Ubiquitin-positive intranuclear inclusions in the granular cell layer of lobule X after 20 weeks of dox induction. (H) FMRpolyG-positive intranuclear inclusions in the granular cell layer of lobule X after 20 weeks of dox induction. All images, 100× magnification.
Figure 4.
Figure 4.
Quantification of ubiquitin-positive intranuclear inclusions in cerebellar lobule X of TRE-90CGG-eGFP/PrP-rtTA mice. (A) The percentage of nuclei containing ubiquitin-positive inclusions after different dox treatment periods. (B) The size of the ubiquitin-positive inclusions after different dox treatment periods. Purple bars represent results from dox-treated mice, and orange bars represent results obtained from mice with an additional washout after dox induction. N = 5–7 mice per group. Significance was determined using a two-tailed t-test with 95% confidence interval, with *P < 0.05; **P < 0.01; ***P < 0.001; N = 5–7 mice per group. Error bars represent SEM.
Figure 5.
Figure 5.
Co-localization of ubiquitin (green) with other components (red) in intranuclear inclusions in the granular cell layer of lobule X of the cerebellum after dox-induced 90CGG RNA expression. Ubiquitin (B, F, J and N) co-localizes (D, H, L and P) in intranuclear inclusions with FMRpolyG (A), Hsp40 (E), Rad23B (I) and 20S subunit of the proteasome (M). Hoechst staining to visualize nuclei (C, G, K and O). All images, ×630 magnification.
Figure 6.
Figure 6.
Deficits in the compensatory eye movements correlate with the presence of inclusions. To determine the functional consequences of expanded CGG repeat-induced inclusions, we tested, in head-fixed awake mice, the compensatory eye movements that are known to depend on an intact vestibulo-cerebellum. (A) The percentage of nuclei in the granular cell layer of cerebellar lobule X containing an inclusion for mice used in the analysis of compensatory eye movements. (B) Sinusoidal rotation of the visual field evoked the OKR, a cerebellum-dependent reflex that minimizes retinal slip. The OKR gain, the ratio of eye-to-stimulus velocity, over a range of frequencies, was compared between mice with and without (ir)reversible inclusions. Whereas gains of 90CCG mice after 8 weeks of dox induction (purple) were not significantly lower than those in controls (black), irreversible inclusions as a result of 20 weeks of exposure to dox (blue) did cause deficits in eye movement performance compared with controls (yellow). In contrast, reversing the presence of inclusions by washout (8 weeks of dox induction followed by 12 weeks of washout, orange) prevented the development of performance deficits. (C) In everyday life, the OKR works in conjunction with the VOR, to maintain a stable image on the retina. To test whether the deficits in the OKR could also affect more natural behaviors, mice were subjected to sinusoidal rotation of the turntable in the light, to evoke the VVOR. All the differences present in the OKR were reproduced, confirming the link of inclusions with behavioral deficits. Insets, colors indicate comparisons with P < 0.05 (repeated-measures ANOVA followed by Tukey's post hoc).

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