Derick G. Wansink

Myotonic dystrophy (DM) is a complex, dominantly inherited, multisystem disorder and the archetypal example of an RNA gain-of-function disease. Unstable expansions of (CTG•CAG)n or (CCTG•CAGG)n repeat tracts in the DMPK and ZNF9 genes cause the two known subtypes of myotonic dystrophy, DM1 and DM2, for which no cure or effective molecular treatment exists. Focus in therapeutic development is currently on toxic, expanded (C/CUG)n RNAs. A series of recent papers provide proof of concept of promising strategies using antisense oligonucleotides or small organic compounds aimed at either complete elimination of expanded (CUG)n RNA transcripts or prevention of detrimental protein binding to thermodynamically stable (C/CUG)n hairpin structures. These developments offer new hope to patients with DM, even though several hurdles still have to be overcome before they can be introduced into clinical practice.

Studies on the myotonic dystrophy protein kinase (DMPK) gene and gene products have thus far mainly concentrated on the fate of length mutation in the (CTG)n repeat at the DNA level and consequences of repeat expansion at the RNA level in DM1 patients and disease models. Surprisingly little is known about the function of DMPK protein products. We demonstrate here that transient expression of one major protein product of the human gene, the hDMPK A isoform with a long tail anchor, results in mitochondrial fragmentation and clustering in the perinuclear region. Clustering occurred in a variety of cell types and was enhanced by an intact tubulin cytoskeleton. In addition to morphomechanical changes, hDMPK A expression induces physiological changes like loss of mitochondrial membrane potential, increased autophagy activity, and leakage of cytochrome c from the mitochondrial intermembrane space accompanied by apoptosis. Truncation analysis using YFP-hDMPK A fusion constructs revealed that the protein's tail domain was necessary and sufficient to evoke mitochondrial clustering behavior. Our data suggest that the expression level of the DMPK A isoform needs to be tightly controlled in cells where the hDMPK gene is expressed. We speculate that aberrant splice isoform expression might be a codetermining factor in manifestation of specific DM1 features in patients.

Myotonic dystrophy type 1 (DM1) is caused by toxicity of an expanded, noncoding (CUG)n tract in DM protein kinase (DMPK) transcripts. According to current evidence the long (CUG)n segment is involved in entrapment of muscleblind (Mbnl) proteins in ribonuclear aggregates and stabilized expression of CUG binding protein 1 (CUGBP1), causing aberrant premRNA splicing and associated pathogenesis in DM1 patients. Here, we report on the use of antisense oligonucleotides (AONs) in a therapeutic strategy for reversal of RNA-gain-of-function toxicity. Using a previously undescribed mouse DM1 myoblast−myotube cell model and DM1 patient cells as screening tools, we have identified a fully 2′-O-methyl-phosphorothioate-modified (CAG)7 AON that silences mutant DMPK RNA expression and reduces the number of ribonuclear aggregates in a selective and (CUG)n-length-dependent manner. Direct administration of this AON in muscle of DM1 mouse models in vivo caused a significant reduction in the level of toxic (CUG)n RNA and a normalizing effect on aberrant premRNA splicing. Our data demonstrate proof of principle for therapeutic use of simple sequence AONs in DM1 and potentially other unstable microsatellite diseases.

Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by an unstable (CTG · CAG)n segment in the 3′ untranslated region of the myotonic dystrophy protein kinase (DMPK) gene. It is commonly accepted that DMPK mRNA-based toxicity is the main contributor to DM1 manifestations; however, not much is known about the significance of the DMPK protein. To appreciate its normal and possible pathobiological role, we analyzed the patterns of DMPK splice isoform expression in mouse tissues. Long membrane–anchored DMPK dominated in heart, diaphragm, and skeletal muscle, whereas short cytosolic isoforms were highly expressed in bladder and stomach. Both isoform types were present in diverse brain regions. DMPK protein was also detectable in cultured myoblasts, myotubes, cortical astrocytes, and related cell lines of neural or muscle origin, but not in hippocampal neurons. This work identifies DMPK as a kinase with pronounced expression in diverse muscle and neural tissues that are affected in DM1. Muscle Nerve, 2009

Trinucleotide instability is a hallmark of degenerative neurological diseases like Huntington's disease, some forms of spinocerebellar ataxia and myotonic dystrophy type 1 (DM1). To investigate the effect of cell type and cell state on the behavior of the DM1 CTG•CAG repeat, we studied a knock-in mouse model for DM1 at different time points during ageing and followed how repeat fate in cells from liver and pancreas is associated with polyploidization and changes in nuclearity after the onset of terminal differentiation.