Correction of GSK3ß at young age prevents muscle pathology in mice with myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is a progressive neuromuscular disease caused by expanded CUG repeats, which misregulate RNA metabolism through several RNA-binding proteins, including CUG-binding protein/CUGBP1 elav-like factor 1 (CUGBP1/CELF1) and muscleblind 1 protein. Mutant CUG repeats elevate CUGBP1 and alter CUGBP1 activity via a glycogen synthase kinase 3ß (GSK3ß)-cyclin D3-cyclin D-dependent kinase 4 (CDK4) signaling pathway. Inhibition of GSK3ß corrects abnormal activity of CUGBP1 in DM1 mice [human skeletal actin mRNA, containing long repeats ( HSALR) model]. Here, we show that the inhibition of GSK3ß in young HSALR mice prevents development of DM1 muscle pathology. Skeletal muscle in 1-yr-old HSALR mice, treated at 1.5 mo for 6 wk with the inhibitors of GSK3, exhibits high fiber density, corrected atrophy, normal fiber size, with reduced central nuclei and normalized grip strength. Because CUG-GSK3ß-cyclin D3-CDK4 converts the active form of CUGBP1 into a form of translational repressor, we examined the contribution of CUGBP1 in myogenesis using Celf1 knockout mice. We found that a loss of CUGBP1 disrupts myogenesis, affecting genes that regulate differentiation and the extracellular matrix. Proteins of those pathways are also misregulated in young HSALR mice and in muscle biopsies of patients with congenital DM1. These findings suggest that the correction of GSK3ß-CUGBP1 pathway in young HSALR mice might have a positive effect on the myogenesis over time.-Wei, C., Stock, L., Valanejad, L., Zalewski, Z. A., Karns, R., Puymirat, J., Nelson, D., Witte, D., Woodgett, J., Timchenko, N. A., Timchenko, L. Correction of GSK3ß at young age prevents muscle pathology in mice with myotonic dystrophy type 1.

Correction of Glycogen Synthase Kinase 3ß in Myotonic Dystrophy 1 Reduces the Mutant RNA and Improves Postnatal Survival of DMSXL Mice.

Myotonic dystrophy type 1 (DM1) is a multisystem neuromuscular disease without cure. One of the possible therapeutic approaches for DM1 is correction of the RNA-binding proteins CUGBP1 and MBNL1, misregulated in DM1. CUGBP1 activity is controlled by glycogen synthase kinase 3ß (GSK3ß), which is elevated in skeletal muscle of patients with DM1, and inhibitors of GSK3 were suggested as therapeutic molecules to correct CUGBP1 activity in DM1. Here, we describe that correction of GSK3ß with a small-molecule inhibitor of GSK3, tideglusib (TG), not only normalizes the GSK3ß-CUGBP1 pathway but also reduces the mutant DMPK mRNA in myoblasts from patients with adult DM1 and congenital DM1 (CDM1). Correction of GSK3ß in a mouse model of DM1 (HSALR mice) with TG also reduces the levels of CUG-containing RNA, normalizing a number of CUGBP1- and MBNL1-regulated mRNA targets. We also found that the GSK3ß-CUGBP1 pathway is abnormal in skeletal muscle and brain of DMSXL mice, expressing more than 1,000 CUG repeats, and that the correction of this pathway with TG increases postnatal survival and improves growth and neuromotor activity of DMSXL mice. These findings show that the inhibitors of GSK3, such as TG, may correct pathology in DM1 and CDM1 via several pathways.

Reduction of Cellular Nucleic Acid Binding Protein Encoded by a Myotonic Dystrophy Type 2 Gene Causes Muscle Atrophy.

Myotonic dystrophy type 2 (DM2) is a neuromuscular disease caused by an expansion of intronic CCTG repeats in the CNBP gene, which encodes a protein regulating translation and transcription. To better understand the role of cellular nucleic acid binding protein (CNBP) in DM2 pathology, we examined skeletal muscle in a new model of Cnbp knockout (KO) mice. This study showed that a loss of Cnbp disturbs myofibrillar sarcomeric organization at birth. Surviving homozygous Cnbp KO mice develop muscle atrophy at a young age. The skeletal muscle phenotype in heterozygous Cnbp KO mice was milder, but they developed severe muscle wasting at an advanced age. Several proteins that control global translation and muscle contraction are altered in muscle of Cnbp KO mice. A search for CNBP binding proteins showed that CNBP interacts with the α subunit of the dystroglycan complex, a core component of the multimeric dystrophin-glycoprotein complex, which regulates membrane stability. Whereas CNBP is reduced in cytoplasm of DM2 human fibers, it is a predominantly membrane protein in DM2 fibers, and its interaction with α-dystroglycan is increased in DM2. These findings suggest that alterations of CNBP in DM2 might cause muscle atrophy via CNBP-mediated translation and via protein-protein interactions affecting myofiber membrane function.

RNA Binding Protein CUGBP1 Inhibits Liver Cancer in a Phosphorylation-Dependent Manner.

Despite intensive investigations, mechanisms of liver cancer are not known. Here, we identified an important step of liver cancer, which is the neutralization of tumor suppressor activities of an RNA binding protein, CUGBP1. The translational activity of CUGBP1 is activated by dephosphorylation at Ser302. We generated CUGBP1-S302A knock-in mice and found that the reduction of translational activity of CUGBP1 causes development of a fatty liver phenotype in young S302A mice. Examination of liver cancer in diethylnitrosamine (DEN)-treated CUGBP1-S302A mice showed these mice develop much more severe liver cancer that is associated with elimination of the mutant CUGBP1. Searching for mechanisms of this elimination, we found that the oncoprotein gankyrin (Gank) preferentially binds to and triggers degradation of dephosphorylated CUGBP1 (de-ph-S302-CUGBP1) or S302A mutant CUGBP1. To test the role of Gank in degradation of CUGBP1, we generated mice with liver-specific deletion of Gank. In these mice, the tumor suppressor isoform of CUGBP1 is protected from Gank-mediated degradation. Consistent with reduction of CUGBP1 in animal models, CUGBP1 is reduced in patients with pediatric liver cancer. Thus, this work presents evidence that de-ph-S302-CUGBP1 is a tumor suppressor protein and that the Gank-UPS-mediated reduction of CUGBP1 is a key event in the development of liver cancer.

Activation of CDK4 Triggers Development of Non-alcoholic Fatty Liver Disease.

The development of non-alcoholic fatty liver disease (NAFLD) is a multiple step process. Here, we show that activation of cdk4 triggers the development of NAFLD. We found that cdk4 protein levels are elevated in mouse models of NAFLD and in patients with fatty livers. This increase leads to C/EBPα phosphorylation on Ser193 and formation of C/EBPα-p300 complexes, resulting in hepatic steatosis, fibrosis, and hepatocellular carcinoma (HCC). The disruption of this pathway in cdk4-resistant C/EBPα-S193A mice dramatically reduces development of high-fat diet (HFD)-mediated NAFLD. In addition, inhibition of cdk4 by flavopiridol or PD-0332991 significantly reduces development of hepatic steatosis, the first step of NAFLD. Thus, this study reveals that activation of cdk4 triggers NAFLD and that inhibitors of cdk4 may be used for the prevention/treatment of NAFLD.