CGG-repeat length and neuropathological and molecular correlates in a mouse model for fragile X-associated tremor/ataxia syndrome

Abstract

The 5'untranslated region (UTR) of the FMR1 gene contains a CGG-repeat, which may become unstable upon transmission to the next generation. When repeat length exceeds 200, the FMR1 gene generally undergoes methylation-mediated transcriptional silencing. The subsequent absence of the gene product Fragile X Mental Retardation Protein (FMRP)causes the mental retardation seen in fragile X patients. A CGG-repeat length between 55 and 200 trinucleotides has been termed the premutation (PM). Predominantly elderly male PM carriers are at risk of developing a progressive neurodegenerative disorder: fragile X-associated tremor/ataxia syndrome (FXTAS). All PM carriers have elevated FMR1 mRNA levels, in spite of slightly decreased FMRP levels. The presence of intranuclear ubiquitin-positive inclusions in many brain regions is a neuropathological hallmark of FXTAS. Studies in humans attempting to correlate neuropathological outcomes with molecular measures are difficult because of the limited availability of tissue. Therefore, we have used the expanded CGG-repeat knock-in mouse model of FXTAS to examine the relationship between the molecular and neuropathological parameters in brain. We present Fmr1 mRNA and Fmrp levels and the presence of intranuclear inclusions at different repeat lengths. Contrary to existing hypotheses, our results suggest that inclusion formation may not depend on the elevation per se of Fmr1 transcript levels in aged CGG mice.

Fig. 1

No ubiquitin-positive inclusions are seen in the colliculus inferior of 72-week-old mice with (CGG)₇₀ (a), while mice with (CGG)₁₀₆ do show ubiquitin-positive inclusions (b) brown, round intranuclear staining). Immunohistochemistry for Fmrp in cortex of (CGG)₇₀ (c) and wt (d) animals gives a similar staining pattern.

Fig. 2

Average fold change of Fmr1 mRNA levels (error bars indicate SDs) in the different (CGG)_n length categories. In these experiments, 13 wt, four (CGG)₇₀, 15 (CGG)_100-150, eight (CGG)_151-200, and 14 (CGG)_{> 200} animals were used. One-way anova revealed a statistically significant overall difference between the categories (F = 6.47, df = 4, p < 0.001). To determine which individual categories differed, Dunnett’s post hoc test was performed. This revealed that only animals with 100-150 CGGs differ significantly from wt animals (fold change mean difference = 1.60, SE = 0.36, p < 0.001, statistical parameters of other categories not shown).

Fig. 3

(a) Average percentages of inclusion-bearing cells in the colliculus inferior and dentate gyrus in the different (CGG)_n length categories. Error bars show SDs. Seven (CGG)_100-150, four (CGG)_151-200, and six (CGG)_{> 200} animals were used. One-way anova revealed a statistically significant difference between the three inclusion-bearing repeat length categories in both the colliculus inferior (F = 8.16, df = 2, p < 0.01) and the dentate gyrus (F = 6.13, df = 2, p = 0.02). Bonferroni-corrected post hoc pair-wise comparisons revealed that inclusion counts did not differ statistically significantly between (CGG)_100-150 and (CGG)_151-200 in the colliculus inferior (mean difference = 0.10, SE = 0.15, p = 1) and the dentate gyrus (mean difference = 0.08, SE = 0.12, p = 0.12). However, inclusion formation in the (CGG)_{> 200} mice differed statistically significantly from (CGG)_100-150 in the colliculus inferior (mean difference = -0.42, SE = 0.11, p < 0.01) and the dentate gyrus (mean difference = -0.30, SE = 0.09, p = 0.02), but not from (CGG)_151-200 (colliculus inferior: mean difference = -0.33, SE = 0.15, p = 0.15, dentate gyrus: mean difference = -0.22, SE = 0.12, p = 0.31). (b) Dentate gyrus of an animal with (CGG)₁₀₆ that does not show intranuclear inclusions, while a mouse with (CGG)₁₃₀ (c) does.

Fig. 4

Ubiquitin-positive inclusions are present in colliculus inferior (a-c) and dentate gyrus (d-f) of mice with (CGG)₁₃₀ and (CGG)₁₈₀, but not in mice with (CGG)_{> 200}.

Fig. 5

Fmrp levels in mouse brain of the different (CGG)_n length categories. (a) Shows an example of a western blot, visualised with the 2F5 antibody against Fmrp, Gapdh was used as a loading control. Approximate (CGG)_n lengths are indicated. (b) Shows average Fmrp levels relative to the average wt level. Error bars represent SDs. A total of three wt, one (CGG)₇₀, 10 (CGG)_100-150, five (CGG)_151-200, and nine (CGG)_{> 200} animals were used for these studies. Note that (CGG)₇₀ has no error bar, as this bar represents one sample. One-way anova revealed that there was a statistically significant difference between (CGG)_n categories (F = 7.02, df = 3, p < 0.01). A Dunnett’s post hoc test was performed to compare Fmrp levels between mice in each repeat category with wt animals. Animals with (CGG)_100-150 didnot show significantly altered Fmrp levels compared to wt mice (%Frmp mean difference = -0.19, SE = 0.11, p = 0.11). Mice with expanded alleles of (CGG)_151-200 (%Frmp mean difference = -0.40, SE = 0.13, p < 0.01) or (CGG)_{> 200} (%Frmp mean difference = -0.44, SE = 0.12, p < 0.01) did have statistically significantly lower Fmrp levels than wt mice. Animals with (CGG)_{> 150} have statistically significantly lowered Fmrp levels (see text for details on statistical analyses).

Fig. 6

Summary of the molecular correlates. Fold change Fmr1 mRNA (left y-axis), % Fmrp and % of inclusion-bearing neurons in colliculus inferior and dentate gyrus (right y-axis) are given. Please refer to the individual graphs for an indication of the variability of the observed data.