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. 2011 Jan 21;6(1):e16417.
doi: 10.1371/journal.pone.0016417.

Long tract of untranslated CAG repeats is deleterious in transgenic mice

Ren-Jun Hsu  1 , Kuang-Ming Hsiao, Min-Jon Lin, Chui-Yen Li, Li-Chun Wang, Luen-Kui Chen, Huichin Pan
Affiliations

Long tract of untranslated CAG repeats is deleterious in transgenic mice

Ren-Jun Hsu et al. PLoS One. .

Abstract

The most frequent trinucleotide repeat found in human disorders is the CAG sequence. Expansion of CAG repeats is mostly found in coding regions and is thought to cause diseases through a protein mechanism. Recently, expanded CAG repeats were shown to induce toxicity at the RNA level in Drosophila and C. elegans. These findings raise the possibility that CAG repeats may trigger RNA-mediated pathogenesis in mammals. Here, we demonstrate that transgenic mice expressing EGFP transcripts with long CAG repeats in the 3' untranslated region develop pathogenic features. Expression of the transgene was directed to the muscle in order to compare the resulting phenotype to that caused by the CUG expansion, as occurs in myotonic dystrophy. Transgenic mice expressing 200, but not those expressing 0 or 23 CAG repeats, showed alterations in muscle morphology, histochemistry and electrophysiology, as well as abnormal behavioral phenotypes. Expression of the expanded CAG repeats in testes resulted in reduced fertility due to defective sperm motility. The production of EGFP protein was significantly reduced by the 200 CAG repeats, and no polyglutamine-containing product was detected, which argues against a protein mechanism. Moreover, nuclear RNA foci were detected for the long CAG repeats. These data support the notion that expanded CAG repeat RNA can cause deleterious effects in mammals. They also suggest the possible involvement of an RNA mechanism in human diseases with long CAG repeats.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Generation of transgenic mice.
(A) Diagram of the transgene constructs. The transgenes contain the gamma-sarcoglycan (GSG) promoter fused with the EGFP coding sequence and followed by SV40 polyadenylation signals (poly A). Insertion of 23 or 200 CAG repeats was made downstream of the EGFP stop codon and before the poly A sequence. Locations of primers (f1, f2, r1 and r2) used for PCR are marked. (B) PCR-based Southern blot analysis. Tail DNA from different founder animals (as indicated by the numbers) generated from the three constructs were PCR-amplified using primers f1 and r2, which generated fragments of 632 bp, 701 bp and 1,232 bp, from CAG0, CAG23 and CAG200 transgenes, respectively. Upper panels, ethidium bromide-stained agarose gels; lower panels, blots of PCR products hybridized with a CAG10 probe.
Figure 2
Figure 2. Transgene expression.
(A) RT-PCR of RNA isolated from different adult tissues. Representative gels showing expression of EGFP in CAG0, CAG23 and CAG200 lines. Amplification of GAPDH was used as an internal control. +, with RT; −, without RT. S, skin; Li, liver; H, heart; L, lung; M, muscle (soleus); T, testis; D, diaphragm; P, pancreas; O, ovary. (B) Northern blot analysis. Representative blot showing skeletal muscle RNA from non-transgenic (NT), CAG0, CAG23 and CAG200 animals hybridized with an EGFP probe or a control GAPDH probe. Relative RNA expression levels (E/G, EGFP divided by GAPDH) are shown below. (C) EGFP protein expression in the transgenic lines determined by western blotting using an anti-EGFP antibody. H, heart; M, muscle; D, diaphragm. Expression of β-tubulin was used as a loading control. (D) EGFP fluorescence was observed using frozen sections from the soleus muscle with a fluorescence microscope. Reduced fluorescence in CAG200 muscle is consistent with the reduced protein expression shown in (C). (E) Detection of polyglutamine-containing protein with a mouse anti-polyglutamine monoclonal antibody (1C2). Protein extracts from the muscle of three mouse lines (50 µg each) and two cell lines transfected with expanded ataxin-3 proteins (54Q, 109Q) (12.5 µg each) were used. Arrowheads indicate the endogenous (45 Kd) and mutant ataxin-3 proteins with 54 or 109 CAG repeats. The position of the wells is indicated to verify the absence of insoluble 1C2-positive proteins. Actin expression was used as a control.
Figure 3
Figure 3. Muscle morphology.
Hematoxylin and eosin-stained paraffin sections of soleus muscle oriented as longitudinal (A–D) or transverse sections (E–H) from 2-month-old non-transgenic (A, E), CAG0 (C), CAG23 (G), and CAG200 (B, D, F, H) animals. Note that the fiber diameters in CAG200 mice (B) are not as uniform as in the control (A) and that multiple rounded nuclei are observed (D). Some nuclei are located internally (arrowheads, F, H) instead of peripherally (C, E, G), and there are signs of split fibers (arrows in F) and angular fibers (arrow in H). (I) Quantification of nuclei (expressed as the nuclei-to-cell ratio). Nuclei and cells in 10 fields in each of four sections from each transgenic line (NT, CAG0-24, CAG23-11, CAG200-32, -62 and one homozygous mouse) were counted using 400× magnification; the average values are presented. *P<0.001. (A–B), 200×; (C–D), 400×; (E–F), 250×; (G–H), 400×.
Figure 4
Figure 4. Histochemical analysis of muscle.
Representative results showing frozen sections of the soleus muscle from 8-month-old non-transgenic (A, E, I), CAG0 -24 (B, F), CAG23-11 (C, G, K) and CAG200-32 (D, H, J, L) animals. (A–D) Staining for succinate dehydrogenase (SDH). Note that normal fibers show very little reaction, whereas “ragged blue” fibers (arrows) are detected in lines CAG200-32 (D) and CAG200-62 (data not shown). (E–J) Staining for NADH-tetrazolium reductases. Note the altered fiber type grouping, moth-eaten patterns, and focal lack of intermyofibrillar network enzyme activity (H, J, arrows) in CAG200 mice compared to clear fiber type distinctions (E–G) and uniform lattices (I) in control animals. (K, L) Staining for ATPase activity at pH 4.3. Type I fibers are black and type II fibers are cream. Type I predominance is seen in CAG200 mice (L). A–H, 160×; I–J, 400×; K–L, 250×.
Figure 5
Figure 5. Phenotypic analysis of transgenic mice.
(A) Grip strength test. Mice were suspended by the forelimbs on a narrow bar and the amount of time before falling was recorded. (B) Cage activity test. CAG200 lines displayed reduced locomotion activity as measured by a grid assay. All data represent the averages from 5 mice that were each tested 3 times (A and B). *, P<0.001; **, P<0.01 (compared to NT, CAG0, or CAG23; F test).
Figure 6
Figure 6. Sperm motility and mitochondrial function.
(A) Counts of total sperm (gray) and motile sperm (white) after a 1-hr incubation at 37°C (n = 6 per group). The average counts are shown below the histogram. Percent sperm motility was determined by dividing the number of motile sperm by total sperm counts. (B) Structure of the sperm tails. Electron microscopy revealed structural defects in the microtubule arrangement of axonemes (loss of one outer doublet; upper right panel, arrows) and in the mitochondria along the midpiece (lower right panel, arrows) in some sperm tails of CAG200 mice. (C) Flow cytometric sorting of rhodamine (Rh123)- and propidium iodide (PI)-stained sperm cells. Horizontal scale, intensity of Rh123; vertical scale, intensity of PI. Note that most sperm cells from CAG200 males are gated with low PI and low Rh123 fluorescence, indicating that they are viable, but have low mitochondrial activity.
Figure 7
Figure 7. Electrophysiology of the muscle.
(A) Action potential. A single action potential was triggered with a depolarizing current of 12 nA at 50-ms duration in NT and CAG200 mice. (B) Phrenic nerve-evoked contracture of isolated diaphragm. In the presence of glucose, significant contracture was induced by a 5-Hz stimulation for 20 min in CAG200, but not NT, CAG0 or CAG23 mice. As a control, all mice produced muscle contracture with a glucose-free Krebs solution using the same stimulation method. +, with glucose; −, without glucose.
Figure 8
Figure 8. Fluorescence in situ hybridization detection of nuclear foci formation.
(A) Frozen muscle sections from CAG0, CAG23 and CAG200 mice were hybridized with a Cy3-(CTG)13 probe (red). The nuclei were counterstained with DAPI (blue). Merged images of red and blue signals are shown. Nuclear foci (arrows) are only detected in CAG200 sections. (B) C2C12 cells transfected with pEGFP-CAG0/58/200 and pEGFP-CTG200 were hybridized with Cy3-(CTG)13 and Cy3-(CAG)13 probes, respectively. Distribution of endogenous MBNL proteins was visualized by immunostaining with an anti-MBNL antibody and a FITC-conjugated secondary antibody (green). Merged images showing superimposition of red and green signals (yellow) demonstrate that MBNL proteins are colocalized with expanded CAG and CUG repeats. Scale bars, 10 µm.
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