Induced expression of expanded CGG RNA causes mitochondrial dysfunction in vivo

Abstract

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.

Keywords: CGG repeat; FXTAS; FXTAS, Fragile X-associated tremor/ataxia syndrome; RNA gain-of-function; TRE, Tet Responsive Element; Tet-On; apoptosis; caspase 3; cytochrome C; dox, doxycycline; eGFP, enhanced green fluorescent protein; gpx, gluthation peroxidase; gpx-1; inducible mouse model; mitochondria; rtTA, reverse tetracycline transactivator.

Figure 1.

New inducible transgenic mouse model. (A) The Tet-On system was used to generate bigenic mice expressing a 11CGG or a 90CGG repeat at the RNA level in all tissues. Expression of rtTA is controlled by the hnRNP promoter on a transgene. Upon dox administration rtTA will be activated and can bind the Tet Responsive Element (TRE) on another transgene, this induces expression of the nCGG repeat at the RNA level and eGFP at the protein level. (B) Genotyping PCR showing the repeat size of 2 × 11 and 2 × 90 CGGs at approximately 390 bp and 630 bp, respectively. (C) 90CGG expressing mice lose weight when compared to 11CGG expressing mice or bigenic mice (TRE-90CGG-eGFP/hnRNP-rtTA) without dox treatment. The straight line with circles shows the weight of mice not treated with dox (n = 7), the dotted line with squares shows the weight of 11CGG expressing mice after 2 mg/ml dox-water treatment (n = 7), and the striped line with triangles show the weight of 90CGG expressing mice after 2 mg/ml dox-water treatment (n = 6). Error bars are +/− SE; * = p < 0.05.

Figure 2.

eGFP expression in different tissues. Representative photomicrographs (40x) of immunohistochemical staining with GFP antibody on heart (A–C), lung (D–F), intestine (G–I), liver (J–L), kidney (M–O), spleen (P–R), and brain (S–U) from no dox control mice (A, D, G, J, M, P, S), TRE-11CCG-eGFP mice (B, E, H, K, N, Q, T), and TRE-90CGG-eGFP mice (C, F, I, L, O, R, U).

Figure 3.

Comparable eGFP RNA and protein expression in the liver of 11CGG and 90CGG mice after 4 d dox treatment. (A) Quantitative RT-PCR on RNA isolated from livers of no dox control mice (white bars; n = 7), TRE-11CGG-eGFP/hnRNP-rtTA mice treated 4 d with dox (gray bars; n = 7), and TRE-90CGG-eGFP/hnRNP-rtTA mice after 4 d dox treatment (black bars; n = 6). 90CGG-eGFP levels are not significantly different from 11CGG-eGFP (p = 0.7) (B) Representative Western blot for eGFP on liver homogenates of no dox control mice and 11CGG or 90CGG expressing mice after 4 d dox treatment with GAPDH as a loading control. (C) Quantification of eGFP protein expression after Western blot on liver homogenates from no dox control mice (white bars; n = 4), TRE-11CGG-eGFP/hnRNP-rtTA mice treated 4 d with dafter 4 d dox treatment (gray bars; n = 8), and TRE-90CGG-eGFP/hnRNP-rtTA mice after 4 d dox treatment (black bars; n = 7). 90CGG-eGFP levels do not significantly differ from the 11CGG-eGFP levels (p = 0.3). Error bars are +/− SE.

Figure 4.

Mitochondrial dysfunction in the liver of 90CGG expressing mice. The color of 90CGG expressing livers is pink and pale (A) compared to the dark reddish-brown color of the liver of control mice (B). Quantitative RT-PCR on RNA isolated from liver of no dox control mice (white bars; n = 7), TRE-11CGG-eGFP/hnRNP-rttA mice treated 4 d with dox (gray bars; n = 7), and TRE-90CGGeGFP/hnRNP-rtTA mice treated 4 d with dox (black bars; n = 6) for oxidative stress markers GPX1 and cytochrome C (C); Error bars are +/− SE; * = p < 0.05; ** = p < 0.01. Representative photomicrographs (40x) of hamatoxylin and eosin (HE) staining (D–F) and cleaved caspase 3 (casp3) immunostaining (G–I) on the livers of no dox controls mice and 11 or 90CGG expressing mice.