Reversal of RNA missplicing and myotonia after muscleblind overexpression in a mouse poly(CUG) model for myotonic dystrophy

Abstract

RNA-mediated pathogenesis is a recently developed disease model that proposes that certain types of mutant genes produce toxic transcripts that inhibit the activities of specific proteins. This pathogenesis model was proposed first for the neuromuscular disease myotonic dystrophy (DM), which is associated with the expansion of structurally related (CTG)(n) and (CCTG)(n) microsatellites in two unrelated genes. At the RNA level, these expansions form stable hairpins that alter the pre-mRNA splicing activities of two antagonistic factor families, the MBNL and CELF proteins. It is unclear which altered activity is primarily responsible for disease pathogenesis and whether other factors and biochemical pathways are involved. Here, we show that overexpression of Mbnl1 in vivo mediated by transduction of skeletal muscle with a recombinant adeno-associated viral vector rescues disease-associated muscle hyperexcitability, or myotonia, in the HSA(LR) poly(CUG) mouse model for DM. Myotonia reversal occurs concurrently with restoration of the normal adult-splicing patterns of four pre-mRNAs that are misspliced during postnatal development in DM muscle. Our results support the hypothesis that the loss of MBNL1 activity is a primary pathogenic event in the development of RNA missplicing and myotonia in DM and provide a rationale for therapeutic strategies designed either to overexpress MBNL1 or inhibit MBNL1 interactions with CUG and CCUG repeat expansions.

Fig. 1.

Expression of Mbnl1 isoforms in skeletal muscle. (A) Distribution of Mbnl1 isoforms in neonatal (P2) and P28 muscle. Ten hindlimb muscle isoforms were identified by RT-PCR followed by cDNA sequencing. Isoforms (color coded) are as follows (in kDa): 43, lime green; 42, black; 41, turquoise; 40, orange; 38, purple; 37, light blue; 36, red; a 36 spliced variant (36*, dark green); 35, yellow; and 32, blue. (B) The major Mbnl1 protein in adult skeletal muscle is 40 kDa. Immunoblot analysis was performed on three skeletal muscles (tibialis anterior, gastrocnemius, and quadriceps), cerebellum, heart, and lung by using either anti-Mbnl1 or anti-Gapdh (loading control).

Fig. 2.

Splicing assay for Mbnl1 isoforms. (A Upper) RNA splicing in HEK293T cells transfected with a Tnnt3 minigene reporter and protein expression plasmids containing GFP, GFP-hnRNP A1 and GFP-CUGBP1, and GFP-Mbnl1 4XC₃H (Mbnl1/40 and Mbnl1/41) and 2XC₃H (Mbnl1/30 and Mbnl1/26) isoforms. (A Lower) An immunoblot of GFP fusion protein expression showing equivalent expression levels for GFP and GFP-fusion proteins after transfection. Protein loading control is Gapdh. PCR primers (arrows) are located in Tnnt3 exons 8 and 9 (open boxes) bordering the alternatively spliced fetal (F) exon (filled box). (B) Phosphorimager quantification of percent fetal exon (FE) exclusion from the PCR data shown in A.

Fig. 3.

Rescue of myotonia after Mbnl1 overexpression. (A) Mbnl1 is overexpressed after AAV2/1-mycMbnl1/41 transduction of TA muscle. TA muscles (23 weeks after injection) were dissected from either uninjected (control) or injected HSA^LR mice (both injected and uninjected muscles are shown) and total protein immunoblotted with anti-MBNL1 antibody. Gapdh is the protein loading control. (B) Distribution of Mbnl1 protein in transverse sections of skeletal muscle. Shown are max-value projections of deconvolved images obtained under identical exposure settings. HSA^LR sections from uninjected (Left) and injected (Right) TA muscles were stained by using the anti-Mbnl1 antibody. (Scale bar: 5 μm.) (C) Myotonia was assessed by electromyography on HSA^LR mice injected in the right TA. The uninjected left TA and gastrocnemius muscles also were tested. The electromyography scale is as follows: 0, no myotonia; 1, occasional myotonic discharge in <50% of needle insertions; 2, myotonic discharge with >50% of insertions; 3, myotonic discharge with nearly all insertions. Uninjected control and injected HSA^LR mice were tested at 4 (purple), 12 (yellow), 23 (turquoise), and 43 (orange) weeks after injection. (D and E) Restoration of Clcn1 protein levels in myofiber membranes after Mbnl1 overexpression. Clcn1 protein levels were detected in uninjected (D) and injected (E) transverse muscle sections at 23 weeks after injection by using an anti-Clcn1 polyclonal antibody (red). DNA distribution is shown by DAPI staining (blue). (Scale bar: 10 μm.) (F and G) Muscle histology (H&E staining) of muscle sections from uninjected (F) and injected (G) TA at 43 weeks after injection.

Fig. 4.

Mbnl1 overexpression promotes adult splicing patterns. RT-PCR splicing assays of uninjected FVB/n and HSA^LR (three mice each) or AAV2/1-mycMbnl1/41 (gene therapy group C, GTC) injected TA at 23 weeks after injection (six mice). Developmentally regulated exons (filled boxes) are either dysregulated in DM (Clcn1, Ldb3/Cypher, Serca1, and Tnnt3) or not affected by the DM expansion mutations (Capzb and Itgb1). Primer positions (arrows) are illustrated below each autoradiograph within constitutively spliced exons (open boxes). The overexpressed mycMbnl1/41 is detectable only in the GTC mice (Bottom).