Molecular, physiological, and motor performance defects in DMSXL mice carrying >1,000 CTG repeats from the human DM1 locus

Abstract

Myotonic dystrophy type 1 (DM1) is caused by an unstable CTG repeat expansion in the 3'UTR of the DM protein kinase (DMPK) gene. DMPK transcripts carrying CUG expansions form nuclear foci and affect splicing regulation of various RNA transcripts. Furthermore, bidirectional transcription over the DMPK gene and non-conventional RNA translation of repeated transcripts have been described in DM1. It is clear now that this disease may involve multiple pathogenic pathways including changes in gene expression, RNA stability and splicing regulation, protein translation, and micro-RNA metabolism. We previously generated transgenic mice with 45-kb of the DM1 locus and >300 CTG repeats (DM300 mice). After successive breeding and a high level of CTG repeat instability, we obtained transgenic mice carrying >1,000 CTG (DMSXL mice). Here we described for the first time the expression pattern of the DMPK sense transcripts in DMSXL and human tissues. Interestingly, we also demonstrate that DMPK antisense transcripts are expressed in various DMSXL and human tissues, and that both sense and antisense transcripts accumulate in independent nuclear foci that do not co-localize together. Molecular features of DM1-associated RNA toxicity in DMSXL mice (such as foci accumulation and mild missplicing), were associated with high mortality, growth retardation, and muscle defects (abnormal histopathology, reduced muscle strength, and lower motor performances). We have found that lower levels of IGFBP-3 may contribute to DMSXL growth retardation, while increased proteasome activity may affect muscle function. These data demonstrate that the human DM1 locus carrying very large expansions induced a variety of molecular and physiological defects in transgenic mice, reflecting DM1 to a certain extent. As a result, DMSXL mice provide an animal tool to decipher various aspects of the disease mechanisms. In addition, these mice can be used to test the preclinical impact of systemic therapeutic strategies on molecular and physiological phenotypes.

Figure 1. Schematic representation of the DMPK/SIX5 region.

Dashed lines represent sense and antisense DMPK transcript. A, B′, C and C′ represent the localization of amplicons used for qRT-PCR. CTCF bs: CTCF binding site; Enh/pro enhancer and promoter region. pA: polyadenylation site for the DMPK sense mRNA The SIX5 start site is indicated with an arrow. The location of the CpG island is indicated in grey.

Figure 2. DMPK expression profiles.

Expression of the human DMPK transgene was studied in various hemizygous DMSXL tissues (A) and muscles (B), in parallel with the endogenous Dmpk mouse gene (C and D) (n = 3). (E) Expression of DMPK in human tissues. Dia., Diaphragm; Sterno., Sternomastoid; Quadri, Quadriceps; TA, Tibialis Anterior; Gastroc. Gastrocnemius. (a.u.): arbitrary units. (F) Expression of DMPK in hemizygous (Hemi.) and homozygous (Homo.) DMSXL tissues. Data are presented as means ± standard deviation.

Figure 3. Expression of sense and antisense DMPK transcripts.

(A–B) DMPK antisense expression profile in 4-month-old DMSXL homozygotes (n = 3) and human control adult tissues (commercial panel) using amplicon B′ located upstream the CAG repeat. (C–D) Comparison of DMPK sense and antisense transcript levels in 4-month-old DMSXL homozygotes (n = 3) and human control tissues. (E–F) Comparison of antisense transcript levels measured in 5′ (before) and in 3′ (after) of the CAG repeat using amplicons B′ and C′ in DMSXL and control human tissues. H, heart; TA, tibialis anterior; Dia, diaphragm; FC, Frontal Cortex; Hemi., Hemizygous; Homo. Homozygous. Data are presented as means ± standard deviation in arbitrary units (a.u.).

Figure 4. Foci distribution in mice carrying CTG expansions.

The distribution and quantification of DMPK RNA foci in various tissues (A) and muscles (B) were compared in 8 month-old homozygous mice with 500/600 CTG (DM300) or 1000/1400 CTG (DMSXL). HL, hind leg; FL foreleg. n is for the number of foci per nucleus. LF: large foci.

Figure 5. Foci accumulation was detected for both sense and antisense DMPK transcripts.

FISH experiments using 5′-Cy3- labeled (CAG)5 or 5′-Cy3- labeled (CTG)5 probes detected respectively, in red, sense and antisense RNA foci in heart (A) and muscle (B) from DMSXL homozygote.

Figure 6. Splicing deregulation in DMSXL mice.

Percentages of alternative exon inclusion in mRNA transcripts were studied by RT-PCR in 2- and 4-month-old mice in tibialis anterior (A–B) and heart (C–D), and in various muscles from 2-month-old mice (E). Results were compared between DMSXL (n = 6) and WT (n = 6). Each bar represents the mean of 6 biological replicates ± SEM. Differences in percentage of alternative exon inclusion between DMSXL and WT mice were determined to be statistically significant by Student's t test, * p<0.05, ** p<0.01, *** p<0.001. 2 m, 2- month-old; 4 m, 4-month-old.

Figure 7. Weight monitoring and follow-up with age.

Weight (in grams, g) was recorded for female (A) and male (B) DMSXL homozygotes (+/+) and WT littermate controls (WT). Differences in body weight between DMSXL and WT mice were determined to be statistically significant by Student's t test, ** p<0.01, *** p<0.001.

Figure 8. Circulating levels of growth-related signaling proteins after fasting.

Significant decreases of IGFBP-3 and insulin levels were observed in 4-month-old homozygous DMSXL. A decrease of IGF-I was also observed but did not reach statistical significance. GH levels were variable but similar between DMSXL and WT. Data are presented as means ± SEM (* p<0.05, ** p<0.01, Student's t test or Mann-Whitney test), n = 12 biological replicates per group.

Figure 9. Histological abnormalities in DMSXL muscles.

(A) Fiber size (cross-sectional area, pixels) was measured in tibialis anterior (TA) and soleus muscles from 4-month-old female WT (grey line) and DMSXL (black line) mice, n = 3 biological replicates in both groups. (B) Fiber types were determined in TA and soleus muscles from WT (grey histograms) and DMSXL (black histograms) mice, n = 3 biological replicates in both groups. (C) Oil Red O staining of muscles section (TA) showed intramyocellular lipid accumulation in DMSXL mice. Data are presented as means ± SEM (* p<0.05, ** p<0.01, *** p<0.001, Chi-square or Student's t tests), n = 4–5 in both groups.

Figure 10. Proteasome activity in DMSXL mice.

Chymotrypsin-like activities of the proteasome were assayed using the fluorogenic peptides LLVY-AMC in 4-month-old female WT and DMSXL mice. Each group contained ten animals and each animal was assayed in triplicate. Histogram bars represent the average activity ± SD for each group (* p<0.05, Student's t test).

Figure 11. Muscle force and strength measurement in DMSXL mice.

(A) Absolute maximal tetanic force was measured in situ for WT and DMSXL female mice between 4 and 6 months of age, as well as muscle mass. The specific maximal tetanic isometric force represented the absolute maximal tetanic isometric force normalized to the muscle mass, n = 8 biological replicates in both groups. (B) Absolute tetanic torque dorsiflexion and plantarflexion in WT and DMSXL mice were determined using a home built dynamometer. Absolute tetanic torque normalized to mouse weight or leg length are also shown, n = 8 for both groups. (C) Grip strength was measured with a grip dynamometer in WT and DMSXL forelegs and hind legs and normalized to body weight. n = 22 biological replicates in both groups. Data are presented as means ± SEM (* p<0.05, ** p<0.01, ANOVA, Student's t test). The mice were analyzed between 4 and 5 months of age. BW: Body weight.

Figure 12. Motor performance of DMSXL mice.

(A) Treadmill test was performed on WT and DMSXL female mice between 4 and 6 months of age. The Critical Speed was calculated from the slope of the regression line, plotting the distance versus the time to exhaustion, n = 9 for both groups. (B) The wheel running test was performed on WT and DMSXL mice between 4 and 6 months of age. The mice were followed over a period of 10 days. The distance, the maximal speed and the time spent running are shown, n = 14 biological replicates for both groups. Data are presented as means ± SEM (* p<0.05, ** p<0.01, Student's t test or Mann-Whitney test).