The expression of CUG repeat RNA in individuals with DM1 induces alterations in two RNA binding proteins; namely, the depletion of MBNL1 and up-regulation of CUGBP1. The role of MBNL1 in disease pathogenesis has been well established through the generation and characterization of MBNL1 knockout mice (23
). Although MBNL1 knockout is sufficient for the development of myotonia and dystrophic muscle histology, MBNL1 knockout mice do not possess a strong muscle wasting phenotype (23
). More recently, transcriptome profiling in MBNL1 knockout mice has revealed widespread changes in transcription, RNA processing and mRNA decay which largely overlap with changes in transgenic mice generated by the expression of CUG250
RNA in the context of the human skeletal actin gene (HSA-CUG250
). Neither MBNL1 knockout mice nor transgenic mice expressing HSA-CUG250
RNA have increased CUGBP1 levels. These studies indicate that MBNL1 can reproduce some features of DM1 independent of alterations in CUGBP1 expression.
So what is the role of CUGBP1, if any, in DM1 pathogenesis? CUGBP1 protein levels are increased in DM1 heart and skeletal muscle (8
), but whether this increase plays a primary role in muscle degeneration or is simply an effect secondary to muscle damage is not known. Several studies using transgenic mice support a pathogenic function of CUGBP1. First, mice expressing DMPK-CUG5
RNA in skeletal muscle had elevated levels of CUGBP1 with no change in MBNL expression or sequestration and showed characteristic DM1 histopathology, myotonia and splicing abnormalities (34
). Second, induction of DMPK-CUG960
RNA in mouse skeletal muscle resulted in a severe muscle wasting phenotype that correlated with an increase in CUGBP1, which has not been mimicked by MBNL1 depletion alone (21
). Third, within 6h following induction of DMPK-CUG960
RNA in mouse cardiac muscle, CUGBP1 levels were increased and pharmacological inhibition of CUGBP1 up-regulation prevented the DM1-like heart phenotype (35
). Fourth, we recently showed that 4-fold overexpression of exogenous CUGBP1 in mouse cardiac tissue reproduced molecular, histopathological and functional changes observed in DM1 mouse models and individuals with DM1 (27
In this paper, we tested the consequence of transgenic expression of exogenous CUGBP1 in adult mouse skeletal muscle. We showed that an 8-fold increase in CUGBP1 resulted in decreased muscle weight and impaired muscle function. By histology, the muscle was reminiscent of DM1 muscle biopsies with a large fraction of myofibers containing centrally located nuclei, pyknotic nuclear clumps and evidence of muscle degeneration. We estimate that less than 10% of the gastrocnemius muscle exhibits regeneration consistent with the low expression of eMHC. In contrast, some of the misregulated splicing changes assayed from gastrocnemius tissue show nearly full reversion to the embryonic patterns. Therefore, we conclude that the large extent of the molecular and phenotypic changes cannot be explained solely based on non-specific responses to muscle regeneration rather than direct effects of CUGBP1 expression.
CUGBP2 and MBNL1 were also increased in MDAFrtTA/TRECUGBP1 mice with an 8-fold increase in CUGBP1. Although it is possible that elevated MBNL1 may have some consequences on the skeletal muscle phenotype in these mice, MDAFrtTA/TRECUGBP1 mice fed a low dose dox diet with only a 2-fold increase in CUGBP1 did not exhibit elevated MBNL1 and yet show histological abnormalities.
A number of alternative splicing events misregulated in DM1 skeletal muscle were shown to be regulated by CUGBP1 and not MBNL1 in transgenic and knockout mice (11
). It is likely that misregulation of these CUGBP1-responsive events in DM1 tissues are a molecular signature of elevated CUGBP1, independent of MBNL1 depletion. In this study, we showed that nine misregulated alternative splicing events identified in DM1 skeletal muscle are also misregulated in the MDAFrtTA/TRECUGBP1 (+dox, 2 weeks) mice with the expression of the embryonic splice variants in adult muscle for all events. Many of the affected transcripts have important functions in skeletal muscle, and their aberrant splicing may have adverse consequences on muscle integrity and function. It has already been reported that alternative splicing of RyR1
exon 70 alters excitation–contraction coupling (19
) and this exon is misregulated in CUGBP1 overexpressing muscle. These data suggest that down-regulation of CUGBP1 in DM1 mouse models expressing DMPK-CUG repeat RNA even in the presence of MBNL1 depletion may ameliorate the disease phenotype.
A progressive loss of inducibility occurred in the MDAFrtTA/TRECUGBP1 mice fed 2 g dox/kg food for 8 weeks, possibly due to the induction of post-transcriptional down-regulation by high CUGBP1 expression or increased doxycycline metabolism by the liver. The inability to maintain long-term induction of CUGBP1 was overcome using a lower doxycycline dosage (0.05 g dox/kg food), but we only achieved a 2-fold up-regulation of CUGBP1 by this method. Although there were some mild histological features in the MDAFrtTA/TRECUGBP1 mice induced with the lower doxycycline dosage diet, there were not other features of DM1 including misregulated alternative splicing. In DM1 tissue and cell cultures, CUGBP1 is stabilized by hyperphosphorylation (10
). It may be possible that in addition to increasing CUGBP1 steady-state levels, hyperphosphorylation alters the protein activity or function and this may be necessary for mediating the effect of CUGBP1 on muscle wasting at lower levels of the protein. Although this very low, stable induction of CUGBP1 over a long time course did not produce a severe muscle wasting phenotype, it may more accurately represents the progressive nature of muscle atrophy in DM1.
The results presented here demonstrate that aberrant expression of CUGBP1 in adult skeletal muscle is pathogenic and produces a phenotype reminiscent of DM1 skeletal muscle. In the future, it will be important to determine the alterations that occur downstream of CUGBP1 up-regulation and which of those events are responsible for the specific phenotypes observed in CUGBP1 overexpressing muscle and individuals with DM1. Although some alternative splicing and translational targets of CUGBP1 have already been identified, global transcriptome profiling will undoubtedly identify new CUGBP1 targets with vital roles in skeletal muscle tissue.