Despite numerous investigations into DM1 pathology, the detailed mechanism underlying this multisystem disease is not well understood, and therapeutic approaches have remained underdeveloped. In this study, we found that the active GSK3β is elevated in skeletal muscle biopsy samples from DM1 patients (Figure ). The increase in GSK3β in DM1 occurs due to accumulation of mutant CUG repeats. This conclusion was made based on studies of two DM1 models: HSALR
mice, expressing CUG repeats in the 3′ UTR of skeletal muscle actin; and CHO double-stable clones expressing Tet-regulated pure CUG repeats. In agreement with our findings, a recent study has shown disruption of AKT/GSK3β signaling in neuronal cells in response to the expression of CUG repeats (40
Our data from the monoclonal CHO cell lines indicate that the elevation of GSK3β is an early event caused by the accumulation of small amounts of mutant CUG repeats. The mutant CUG repeats in this cell model are detectable by FISH and Northern blot assays mainly at 7 hours after Dox addition (Supplemental Figure 1 and ref. 38
). We found that GSK3β increased within 2 hours after induction of CUG914
expression, suggesting that even small amounts of CUG repeats, which are difficult to detect by FISH assay, are sufficient to elevate GSK3β. The mechanism of CUG repeat–dependent elevation of active GSK3β involves the stabilization of GSK3β through an increase in its activity. This conclusion is supported by results indicating that inhibition of GSK3β activity by lithium corrected GSK3β protein levels (Figure E). Since the stabilization of GSK3β correlates with an increase in its autophosphorylation, the most likely mechanism is that CUG repeats first increase activity of GSK3β, and this in turn activates GSK3β phosphorylation, stabilizing GSK3β. How might CUG repeats activate GSK3β? Since RNA-binding activity of GSK3β has not been reported, it is clear that there is a pathway mediator that is activated by CUG repeats. Protein factors or toxic peptides, synthesized from expanded CUG RNA through an AUG-independent mechanism, might trigger the elevation of GSK3β activity (41
). These factors involved in activating a CUG-mediated increase in GSK3β remain to be identified.
One of the most important results of this study is the identification of GSK3β as a crucial signaling molecule associated with the reduction of cyclin D3 and muscle weakness and myotonia in HSALR mice. Whereas initial reports had not described progressive muscle weakness and wasting in these mice, we found reproducible and statistically significant reduction of muscle strength in 3-month- and 6-month-old HSALR mice. Our data show that muscle in the 1-month-old HSALR mice was characterized by an increased number of nuclei located beneath the basal lamina and by an increased number of small-size myofibers (Figure ). In agreement with this, the levels of marker of satellite cells Pax-7 were significantly increased in skeletal muscle of 1-month-old HSALR mice. The number of newly activated proliferating satellite cells also increased in young HSALR mice. These data suggest that in young HSALR mice, muscle is actively regenerating due to activation and proliferation of satellite cells. As a result, muscle regeneration prevents the development of muscle weakness. However, muscles in adult (6-month-old) HSALR mice had a reduced number of nuclei located beneath the basal lamina, reduced numbers of Pax-7–positive satellite cells, and reduced numbers of myofibers. These data suggest that muscle in adult HSALR mice cannot efficiently regenerate, and, as a result, myofibers are degenerating.
Since lithium has other targets in addition to GSK3β, we used a highly selective inhibitor of GSK3β, TDZD-8. Similar to lithium, TDZD-8 reduced muscle weakness in HSALR mice. This improvement in muscle strength was accompanied by a correction of GSK3β and cyclin D3 levels. The treatment of mice with TDZD-8 was more efficient than that with lithium. We observed an improvement in muscle strength in HSALR mice treated with lithium 2 weeks after initiation of treatment. However, the beneficial effect of TDZD-8 on muscle strength in these mice was observed within only 2 days after initiation of treatment.
In addition to improving skeletal muscle strength, the lithium and TDZD-8 treatments also reduced myotonia. The mechanism responsible for the correction of myotonia by the lithium and TDZD-8 remains to be investigated. It has been suggested that myotonia in DM1 is due to mis-splicing of the chloride channel (CLCN1
). Since this splicing of CLCN1
is regulated by two proteins, MBNL1 and CUGBP1, we suggest that activation of CUGBP1 by cyclin D3/CDK4 may improve not only the translational activity of CUGBP1, but also splicing activity, reducing myotonia. We cannot exclude the possibility that lithium and TDZD-8 treatments also have a positive effect on MBNL1 splicing activity; however, this possibility is unlikely, because GSK3β elevation occurs prior to buildup of CUG foci that sequester MBNL1 (Figure , C and D, Supplemental Figure 1, and refs. 22
One of the important questions to be answered is, What is the most appropriate timing for treatment of DM1 with GSK3β inhibitors? We found that TDZD-8 treatment of young (6 weeks old) HSALR mice for 1 week improved their grip strength by 10.9% (Supplemental Figure 4). We tested grip strength in the same mice at 3 months of age and found that their grip strength was 9.2% higher than in the age- and sex-matched untreated HSALR mice. These data suggest that the inhibition of GSK3β in HSALR mice at a young age, when they show an insignificant reduction in grip strength, delays development of muscle weakness at 3 months of age. Use of inducible mouse models with temporary expression of CUG repeats at a young age, with simultaneous treatment with lithium or other potent inhibitors of GSK3β such as TDZD-8, would be a good model for determining the best timing of treatment of these mice.
In conclusion, this study shows that the mutant CUG repeats elevate active GSK3β in DM1 muscle and that inhibition of GSK3β with lithium or TDZD-8 improves muscle strength and reduces myotonia in the DM1 mouse model. Results in this study and the data described in the literature suggest that CUG repeats cause the disease through several pathways: (a) elevation of active CUGBP1 due to an increase of its stability, causing mis-regulation of translation, splicing, and stability of mRNAs controlled by CUGBP1; (b) reduction of MBNL1 due to sequestration by foci, causing reduction of splicing of MBNL1-regulated mRNAs; and (c) mis-regulation of signaling in DM1 cells, in particular GSK3β signaling, which leads to elevation of the inactive form of CUGBP1 in DM1 muscle. The positive effect of lithium and TDZD-8 on skeletal muscle function in HSALR mice suggests that lithium or other GSK3 antagonists that correct the GSK3β/cyclin D3/CUGBP1 pathway might be candidates for DM1 therapy.