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. 2017 Jun 1;26(11):2133-2145.
doi: 10.1093/hmg/ddx108.

Selective rescue of heightened anxiety but not gait ataxia in a premutation 90CGG mouse model of Fragile X-associated tremor/ataxia syndrome

Hoanna Castro  1 , Emre Kul  1 , Ronald A M Buijsen  2 , Lies-Anne W F M Severijnen  2 , Rob Willemsen  2 , Renate K Hukema  2 , Oliver Stork  1   3 , Mónica Santos  1
Affiliations

Selective rescue of heightened anxiety but not gait ataxia in a premutation 90CGG mouse model of Fragile X-associated tremor/ataxia syndrome

Hoanna Castro et al. Hum Mol Genet. .

Abstract

A CGG-repeat expansion in the premutation range in the Fragile X mental retardation 1 gene (FMR1) has been identified as the genetic cause of Fragile X-associated tremor/ataxia syndrome (FXTAS), a late-onset neurodegenerative disorder that manifests with action tremor, gait ataxia and cognitive impairments. In this study, we used a bigenic mouse model, in which expression of a 90CGG premutation tract is activated in neural cells upon doxycycline administration-P90CGG mouse model. We, here, demonstrate the behavioural manifestation of clinically relevant features of FXTAS patients and premutation carrier individuals in this inducible mouse model. P90CGG mice display heightened anxiety, deficits in motor coordination and impaired gait and represent the first FXTAS model that exhibits an ataxia phenotype as observed in patients. The behavioural phenotype is accompanied by the formation of ubiquitin/FMRpolyglycine-positive intranuclear inclusions, as another hallmark of FXTAS, in the cerebellum, hippocampus and amygdala. Strikingly, upon cessation of transgene induction the anxiety phenotype of mice recovers along with a reduction of intranuclear inclusions in dentate gyrus and amygdala. In contrast, motor function deteriorates further and no reduction in intranuclear inclusions can be observed in the cerebellum. Our data thus demonstrate that expression of a 90CGG premutation expansion outside of the FMR1 context is sufficient to evoke an FXTAS-like behavioural phenotype. Brain region-specific neuropathology and (partial) behavioural reversibility make the inducible P90CGG a valuable mouse model for testing pathogenic mechanisms and therapeutic intervention methods.

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Figures

Figure 1
Figure 1
Motor Impairments in the inducible P90CGGDOX+ animals mimic FXTAS ataxic gait. Inducible P90CGG animals were given a 12-week period of doxycycline administration, starting after weaning, and tested in a behavioural battery that includes evaluation of motor function in the rotarod and footprint pattern. (A) In the training sessions, P90CGGDOX+ animals showed a worse performance than P90CGGDOX- controls, which was overcome with overtraining and P90CGGDOX+ animals were able to reach the same performance levels as controls (repeated measures two-way ANOVA, P90CGGDOX-n = 11, P90CGGDOX+n = 17). In the fourth day, the same animals were tested for (B) motor learning and (C) motor coordination and for both paradigms P90CGGDOX+ animals showed deficits in performance as compared with controls (Student’s t-test). (D) Footprints were obtained for all animals (P90CGGDOX-n = 11, P90CGGDOX+n = 17) and used to analyse the gait pattern by measuring (d1) frontpaws width, (d2) hindpaws width, (d3) stride length and (d4) uniformity of step alternation. Representative footprint patterns of P90CGG animals treated (E) without and (F) with DOX. P90CGGDOX+ animals showed an increase in the width of uniformity of step alternation as compared with P90CGGDOX- control animals (J) and no differences in the other parameters analysed (G–I) (Student’s t-test). P90CGG.DOX-, animals administered 5% saccharose; P90CGG.DOX+, animals administered doxycycline. Repeated measures two-way ANOVA, genotype effect, #P < 0.05; Student’s t-test, *P < 0.05, **P < 0.01.
Figure 2
Figure 2
Inducible P90CGG mouse model exhibit psychiatric-like features associated with FXTAS and premutation carriers. Inducible P90CGG animals were given a 12-week period of doxycycline administration, starting after weaning, and then tested in a battery of tests that include emotional and cognitive assessment. In the open field (P90CGGDOX-n = 11, P90CGGDOX+n = 17), P90CGGDOX+ and control animals showed (A) a similar total distance travelled in the arena (repeated measures two-way ANOVA). (B) P90CGGDOX+ animals exhibited heighten anxiety levels, as given by a reduction in the percentage of time spent in the centre of the open field arena, as compared with P90CGGDOX- controls (repeated measures two-way ANOVA). (C, D) Home cage spontaneous locomotor activity was assessed in a 24-h period for three consecutive days. P90CGGDOX+ animals showed hyperactivity specifically during dark phase of the day, which corresponds to the active period of rodents (repeated measures two-way ANOVA, P90CGGDOX-n = 8, P90CGGDOX+n = 14). Mice were tested for learning and memory using an auditory fear-conditioning paradigm. No differences were observed between P90CGGDOX+ and P90CGGDOX- in the (E) habituation, (F) fear-conditioning training, (G) context fear memory or (H) cue fear memory (repeated measures two-way ANOVA, P90CGGDOX-n = 14, P90CGGDOX+n = 15). P90CGG.DOX-, animals administered 5% saccharose; P90CGG.DOX+, animals administered doxycycline. Repeated measures two-way ANOVA, genotype effect, #P < 0.05; time/training/CS effect, §P < 0.05, §§P < 0.01; time × genotype effect, &P < 0.05; Student’s t-test, **P < 0.01.
Figure 3
Figure 3
No motor impairments or anxiety alterations were observed in the inducible P11CGG mouse model. Inducible P11CGG animals were given a 12-week period of doxycycline administration, starting after weaning, and then tested in the behavioural paradigms for which P90CGGDOX+ animals showed differences as compared with their P90CGGDOX- controls. The objective is to distinguish whether P90CGGDOX+ behavioural alterations are indeed due to CGG repeat size or whether RNA load could account for the observed differences. (A) In the training sessions of the rotarod paradigm P11CGGDOX+ acquire the task as well as P11CGGDOX- animals (repeated measures two-way ANOVA, P11CGGDOX-n = 11, P11CGGDOX+n = 12). (B) When testing motor learning, at 36 rpm P11CGGDOX+ animals showed a reduction in the latency to fall off the rod, as compared with P11CGGDOX- animals (Student’s t-test). (C) No differences in motor coordination were observed between P11CGG treatment groups (Student’s t-test). Analysis of footprint patterns showed no differences in gait between P11CGGDOX+ and P11CGGDOX- animals when (D) forepaws width, (E) hindpaws width, (F) stride length and (G) uniformity of step alternation parameters were measured (Student’s t-test, P11CGGDOX-n = 11, P11CGGDOX+n = 12). No differences were observed in the performance of P11CGGdox+ and P11CGGDOX- animals in the (H) total distance travelled and (I) percentage of time spent in the centre of the arena of the open field (repeated measures two-way ANOVA, P11CGGDOX-n = 11, P11CGGDOX+n = 12). (J, K) Assessment of home cage activity in a 24-h period for three consecutive days showed no differences in spontaneous locomotor activity between P11CGGDOX+/DOX- treatment groups (repeated measures two-way ANOVA, P11CGGDOX-n = 11, P11CGGDOX+n = 12). P11CGG.DOX-, animals administered 5% saccharose; P11CGG.DOX+, animals administered doxycycline. Repeated measures two-way ANOVA, training/time effect, §§P < 0.01, §§§P < 0.001; time × genotype effect, &&&P < 0.001; Student’s t-test, ***P < 0.001.
Figure 4
Figure 4
Rescue of heightened anxiety, but not of gait ataxia in P90CGGDOX+ after shutdown of transgene expression. Inducible P90CGG animals were given a 12-week washout period after initial 12 + 3 weeks of DOX administration to assess whether behavioural rescue was possible (P90CGGDOX-.WOn = 8, P90CGGDOX+.WOn = 13). Footprint patterns were obtained for P90CGG animals after the 12-week washout period and analysed for (A) forepaws width, (B) hindpaws width, (C) stride length and (D) uniformity of step alternation parameters. P90CGGDOX+.WO animals showed alterations in gait after the washout period (Student’s t-test). Interestingly, no differences between P90CGGDOX+.WO and P90CGGDOX-.WO controls were observed in (E) the percentage of time spent in the centre of the open field (repeated measures two-way ANOVA) or (F) in the percentage of activity in the lit compartment of a light–dark box (Student’s t-test) showing a rescue of heightened anxiety levels. P90CGG.DOX-, animals administered 5% saccharose; P90CGG.DOX+, animals administered doxycycline; WO, washout period. Student’s t-test, **P < 0.01.
Figure 5
Figure 5
Formation and clearance of ubiquitin-positive intranuclear inclusions in the inducible P90CGG mouse model is brain region-dependent. Representative pictures of ubiquitin-positive intranuclear inclusions in (A, B) cerebellum lobule X (10× and 100×, respectively), (C) amygdala (10×), (D) mainly in basolateral amygdala (100×) and (E) hippocampus (10×), (F) mainly in CA3 sub-region (100×). (G) Quantification of ubiquitin-positive intranuclear inclusions in P90CGG animals given a 12 + 3 weeks of doxycycline administration, starting after weaning (P90CGGDOX+n = 7/9), and in animals treated with an additional 12-week washout period (P90CGGDOX+.WOn = 7/10). After a 12-week washout period intranuclear inclusions are cleared mostly in the amygdala and hippocampus, but not in the cerebellum (Student’s t-test). P90CGG.DOX+, animals administered doxycycline; P90CGG.DOX+.WO, doxycycline treated animals submitted to a washout period. BLA, basolateral amygdala, CA1, cornus Ammon 1, CA3, cornus Ammon 3, CeA, central amygdala, DG, dentate gyrus, lb X, lobule X of the cerebellum. Student’s t-test, *P < 0.05.
Figure 6
Figure 6
The inducible P90CGG mouse model does not exhibit motor impairments after a shorter, 8-week period of transgene expression. Inducible P90CGG animals were given an 8-week period of doxycycline administration, starting after weaning, and tested in tests that reflect motor and anxiety performance (P90CGGDOXs-n = 14, P90CGGDOXs+n = 15). In the rotarod, no differences in motor performance were found between P90CGGDOXs+ and P90CGGDOXs- control animals in the latency to fall off the rod at (A) the training sessions (repeated measures two-way ANOVA) or when testing for (B) motor learning or (C) motor coordination (Student’s t-test). Footprints were obtained for all animals and used to analyse the gait pattern by measuring (D) forepaws width, (E) hindpaws width, (F) stride length and (G) uniformity of step alternation. No differences were observed between P90CGGDOXs treatment groups for any of the gait parameters analysed (Student’s t-test). In the open field, no differences were observed in (H) the total distance travelled or (I) the percentage of time spent in the centre of the arena (repeated measures two-way ANOVA). Shutdown 90CGG transgene at 8 weeks is sufficient to rescue motor and anxiety levels at this time point. P90CGG.DOXs-, animals administered 5% saccharose; P90CGG.DOXs+, animals administered doxycycline for 8 weeks. Repeated measures two-way ANOVA, genotype effect, #P < 0.05; time effect, &P < 0.05.
Figure 7
Figure 7
Early intervention delays but does not prevent gait impairments in the inducible P90CGG mouse model. Inducible P90CGG animals of the 8-week DOX group were given a 12-week washout period and assessed for motor performance (P90CGGDOXs-.WOn = 8, P90CGGDOXs+.WOn = 9). Footprint patterns were obtained for P90CGG 8-week DOX animals after the 12-week washout period and analysed for gait parameters. After the washout period, P90CGGDOXs+.WO animals show (A) a reduction in stride length and (B) a trend to significant reduction in forepaws width as compared with P90CGGDOXs-.WO. (C, D) Representative pictures of FMRpolyG-positive inclusions in the cerebellum lobule X of P90CGGDOXs+ animals (P90CGGDOX+n = 4, P90CGGDOXs+.WOn = 7) at 10× and 100× magnification, respectively. (E) Quantification of FMRpolyG-positive intranuclear inclusions in cerebellum lobule X of P90CGGDOXs+ and P90CGGDOXs+.WO animals. P90CGG.DOXs-, animals administered 5% saccharose, P90CGG.DOXs+, animals administered doxycycline for 8 weeks; WO, animals submitted to a washout period. Student’s t-test, *P < 0.05.
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