MBNL-dependent impaired development within the neuromuscular system in myotonic dystrophy type 1.

AIMS: Myotonic dystrophy type I (DM1) is one of the most frequent muscular dystrophies in adults. Although DM1 has long been considered mainly a muscle disorder, growing evidence suggests the involvement of peripheral nerves in the pathogenicity of DM1 raising the question of whether motoneurons (MNs) actively contribute to neuromuscular defects in DM1. METHODS: By using micropatterned 96-well plates as a coculture platform, we generated a functional neuromuscular model combining DM1 and muscleblind protein (MBNL) knock-out human-induced pluripotent stem cells-derived MNs and human healthy skeletal muscle cells. RESULTS: This approach led to the identification of presynaptic defects which affect the formation or stability of the neuromuscular junction at an early developmental stage. These neuropathological defects could be reproduced by the loss of RNA-binding MBNL proteins, whose loss of function in vivo is associated with muscular defects associated with DM1. These experiments indicate that the functional defects associated with MNs can be directly attributed to MBNL family proteins. Comparative transcriptomic analyses also revealed specific neuronal-related processes regulated by these proteins that are commonly misregulated in DM1. CONCLUSIONS: Beyond the application to DM1, our approach to generating a robust and reliable human neuromuscular system should facilitate disease modelling studies and drug screening assays.

Deciphering the Complex Molecular Pathogenesis of Myotonic Dystrophy Type 1 through Omics Studies.

Omics studies are crucial to improve our understanding of myotonic dystrophy type 1 (DM1), the most common muscular dystrophy in adults. Employing tissue samples and cell lines derived from patients and animal models, omics approaches have revealed the myriad alterations in gene and microRNA expression, alternative splicing, 3' polyadenylation, CpG methylation, and proteins levels, among others, that contribute to this complex multisystem disease. In addition, omics characterization of drug candidate treatment experiments provides crucial insight into the degree of therapeutic rescue and off-target effects that can be achieved. Finally, several innovative technologies such as single-cell sequencing and artificial intelligence will have a significant impact on future DM1 research.

A low absolute number of expanded transcripts is involved in myotonic dystrophy type 1 manifestation in muscle.

Muscular manifestation of myotonic dystrophy type 1 (DM1), a common inheritable degenerative multisystem disorder, is mainly caused by expression of RNA from a (CTG·CAG)n-expanded DM1 locus. Here, we report on comparative profiling of expression of normal and expanded endogenous or transgenic transcripts in skeletal muscle cells and biopsies from DM1 mouse models and patients in order to help us in understanding the role of this RNA-mediated toxicity. In tissue of HSA(LR) mice, the most intensely used 'muscle-only' model in the DM1 field, RNA from the α-actin (CTG)250 transgene was at least 1000-fold more abundant than that from the Dmpk gene, or the DMPK gene in humans. Conversely, the DMPK transgene in another line, DM500/DMSXL mice, was expressed ∼10-fold lower than the endogenous gene. Temporal regulation of expanded RNA expression differed between models. Onset of expression occurred remarkably late in HSA(LR) myoblasts during in vitro myogenesis whereas Dmpk or DMPK (trans)genes were expressed throughout proliferation and differentiation phases. Importantly, quantification of absolute transcript numbers revealed that normal and expanded Dmpk/DMPK transcripts in mouse models and DM1 patients are low-abundance RNA species. Northern blotting, reverse transcriptase-quantitative polymerase chain reaction, RNA-sequencing and fluorescent in situ hybridization analyses showed that they occur at an absolute number between one and a few dozen molecules per cell. Our findings refine the current RNA dominance theory for DM1 pathophysiology, as anomalous factor binding to expanded transcripts and formation of soluble or insoluble ribonucleoprotein aggregates must be nucleated by only few expanded DMPK transcripts and therefore be a small numbers game.

Myotonic dystrophy RNA toxicity alters morphology, adhesion and migration of mouse and human astrocytes.

Brain dysfunction in myotonic dystrophy type 1 (DM1), the prototype of toxic RNA disorders, has been mainly attributed to neuronal RNA misprocessing, while little attention has been given to non-neuronal brain cells. Here, using a transgenic mouse model of DM1 that expresses mutant RNA in various brain cell types (neurons, astroglia, and oligodendroglia), we demonstrate that astrocytes exhibit impaired ramification and polarization in vivo and defects in adhesion, spreading, and migration. RNA-dependent toxicity and phenotypes are also found in human transfected glial cells. In line with the cell phenotypes, molecular analyses reveal extensive expression and accumulation of toxic RNA in astrocytes, which result in RNA spliceopathy that is more severe than in neurons. Astrocyte missplicing affects primarily transcripts that regulate cell adhesion, cytoskeleton, and morphogenesis, and it is confirmed in human brain tissue. Our findings demonstrate that DM1 impacts astrocyte cell biology, possibly compromising their support and regulation of synaptic function.

Integrative Cell Type-Specific Multi-Omics Approaches Reveal Impaired Programs of Glial Cell Differentiation in Mouse Culture Models of DM1.

Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by a non-coding CTG repeat expansion in the DMPK gene. This mutation generates a toxic CUG RNA that interferes with the RNA processing of target genes in multiple tissues. Despite debilitating neurological impairment, the pathophysiological cascade of molecular and cellular events in the central nervous system (CNS) has been less extensively characterized than the molecular pathogenesis of muscle/cardiac dysfunction. Particularly, the contribution of different cell types to DM1 brain disease is not clearly understood. We first used transcriptomics to compare the impact of expanded CUG RNA on the transcriptome of primary neurons, astrocytes and oligodendrocytes derived from DMSXL mice, a transgenic model of DM1. RNA sequencing revealed more frequent expression and splicing changes in glia than neuronal cells. In particular, primary DMSXL oligodendrocytes showed the highest number of transcripts differentially expressed, while DMSXL astrocytes displayed the most severe splicing dysregulation. Interestingly, the expression and splicing defects of DMSXL glia recreated molecular signatures suggestive of impaired cell differentiation: while DMSXL oligodendrocytes failed to upregulate a subset of genes that are naturally activated during the oligodendroglia differentiation, a significant proportion of missplicing events in DMSXL oligodendrocytes and astrocytes increased the expression of RNA isoforms typical of precursor cell stages. Together these data suggest that expanded CUG RNA in glial cells affects preferentially differentiation-regulated molecular events. This hypothesis was corroborated by gene ontology (GO) analyses, which revealed an enrichment for biological processes and cellular components with critical roles during cell differentiation. Finally, we combined exon ontology with phosphoproteomics and cell imaging to explore the functional impact of CUG-associated spliceopathy on downstream protein metabolism. Changes in phosphorylation, protein isoform expression and intracellular localization in DMSXL astrocytes demonstrate the far-reaching impact of the DM1 repeat expansion on cell metabolism. Our multi-omics approaches provide insight into the mechanisms of CUG RNA toxicity in the CNS with cell type resolution, and support the priority for future research on non-neuronal mechanisms and proteomic changes in DM1 brain disease.

Suppression of Mutant Protein Expression in SCA3 and SCA1 Mice Using a CAG Repeat-Targeting Antisense Oligonucleotide.

Spinocerebellar ataxia type 3 (SCA3) and type 1 (SCA1) are dominantly inherited neurodegenerative disorders that are currently incurable. Both diseases are caused by a CAG-repeat expansion in exon 10 of the Ataxin-3 and exon 8 of the Ataxin-1 gene, respectively, encoding an elongated polyglutamine tract that confers toxic properties to the resulting proteins. We have previously shown lowering of the pathogenic polyglutamine protein in Huntington's disease mouse models using (CUG)7, a CAG repeat-targeting antisense oligonucleotide. Here we evaluated the therapeutic capacity of (CUG)7 for SCA3 and SCA1, in vitro in patient-derived cell lines and in vivo in representative mouse models. Repeated intracerebroventricular (CUG)7 administration resulted in a significant reduction of mutant Ataxin-3 and Ataxin-1 proteins throughout the brain of SCA3 and SCA1 mouse models, respectively. Furthermore, in both a SCA3 patient cell line and the MJD84.2 mouse model, (CUG)7 induced formation of a truncated Ataxin-3 protein species lacking the polyglutamine stretch, likely arising from (CUG)7-mediated exon 10 skipping. In contrast, skipping of exon 8 of Ataxin-1 did not significantly contribute to the Ataxin-1 protein reduction observed in (CUG)7-treated SCA1154Q/2Q mice. These findings support the therapeutic potential of a single CAG repeat-targeting AON for the treatment of multiple polyglutamine disorders.

The expanded CAG repeat in the huntingtin gene as target for therapeutic RNA modulation throughout the HD mouse brain.

The aim of these studies was to demonstrate the therapeutic capacity of an antisense oligonucleotide with the sequence (CUG)7 targeting the expanded CAG repeat in huntingtin (HTT) mRNA in vivo in the R6/2 N-terminal fragment and Q175 knock-in Huntington's disease (HD) mouse models. In a first study, R6/2 mice received six weekly intracerebroventricular infusions with a low and high dose of (CUG)7 and were sacrificed 2 weeks later. A 15-60% reduction of both soluble and aggregated mutant HTT protein was observed in striatum, hippocampus and cortex of (CUG)7-treated mice. This correction at the molecular level resulted in an improvement of performance in multiple motor tasks, increased whole brain and cortical volume, reduced levels of the gliosis marker myo-inositol, increased levels of the neuronal integrity marker N-aceyl aspartate and increased mRNA levels of the striatal marker Darpp-32. These neuroanatomical and neurochemical changes, together with the improved motor performance, suggest that treatment with (CUG)7 ameliorates basal ganglia dysfunction. The HTT-lowering was confirmed by an independent study in Q175 mice using a similar (CUG)7 AON dosing regimen, further demonstrating a lasting reduction of mutant HTT protein in striatum, hippocampus and cortex for up to 18 weeks post last infusion along with an increase in motor activity. Based on these encouraging results, (CUG)7 may thus offer an interesting alternative HTT-lowering strategy for HD.

Intracellular Distribution and Nuclear Activity of Antisense Oligonucleotides After Unassisted Uptake in Myoblasts and Differentiated Myotubes In Vitro.

Clinical efficacy of antisense oligonucleotides (AONs) for the treatment of neuromuscular disorders depends on efficient cellular uptake and proper intracellular routing to the target. Selection of AONs with highest in vitro efficiencies is usually based on chemical or physical methods for forced cellular delivery. Since these methods largely bypass existing natural mechanisms for membrane passage and intracellular trafficking, spontaneous uptake and distribution of AONs in cells are still poorly understood. Here, we report on the unassisted uptake of naked AONs, so-called gymnosis, in muscle cells in culture. We found that gymnosis works similarly well for proliferating myoblasts as for terminally differentiated myotubes. Cell biological analyses combined with microscopy imaging showed that a phosphorothioate backbone promotes efficient gymnosis, that uptake is clathrin mediated and mainly results in endosomal-lysosomal accumulation. Nuclear localization occurred at a low level, but the gymnotically delivered AONs effectively modulated the expression of their nuclear RNA targets. Chloroquine treatment after gymnotic delivery helped increase nuclear AON levels. In sum, we demonstrate that gymnosis is feasible in proliferating and non-proliferating muscle cells and we confirm the relevance of AON chemistry for uptake and intracellular trafficking with this method, which provides a useful means for bio-activity screening of AONs in vitro.

Cell membrane integrity in myotonic dystrophy type 1: implications for therapy.

Myotonic Dystrophy type 1 (DM1) is a multisystemic disease caused by toxic RNA from a DMPK gene carrying an expanded (CTG•CAG)n repeat. Promising strategies for treatment of DM1 patients are currently being tested. These include antisense oligonucleotides and drugs for elimination of expanded RNA or prevention of aberrant binding to RNP proteins. A significant hurdle for preclinical development along these lines is efficient systemic delivery of compounds across endothelial and target cell membranes. It has been reported that DM1 patients show elevated levels of markers of muscle damage or loss of sarcolemmal integrity in their serum and that splicing of dystrophin, an essential protein for muscle membrane structure, is abnormal. Therefore, we studied cell membrane integrity in DM1 mouse models commonly used for preclinical testing. We found that membranes in skeletal muscle, heart and brain were impermeable to Evans Blue Dye. Creatine kinase levels in serum were similar to those in wild type mice and expression of dystrophin protein was unaffected. Also in patient muscle biopsies cell surface expression of dystrophin was normal and calcium-positive fibers, indicating elevated intracellular calcium levels, were only rarely seen. Combined, our findings indicate that cells in DM1 tissues do not display compromised membrane integrity. Hence, the cell membrane is a barrier that must be overcome in future work towards effective drug delivery in DM1 therapy.

Design and analysis of effects of triplet repeat oligonucleotides in cell models for myotonic dystrophy.

Myotonic dystrophy type 1 (DM1) is caused by DM protein kinase (DMPK) transcripts containing an expanded (CUG)n repeat. Antisense oligonucleotide (AON)-mediated suppression of these mutant RNAs is considered a promising therapeutic strategy for this severe disorder. Earlier, we identified a 2'-O-methyl (2'-OMe) phosphorothioate (PT)-modified (CAG)7 oligo (PS58), which selectively silences mutant DMPK transcripts through recognition of the abnormally long (CUG)n tract. We present here a comprehensive collection of triplet repeat AONs and found that oligo length and nucleotide chemistry are important determinants for activity. For significant reduction of expanded DMPK mRNAs, a minimal length of five triplets was required. 2'-O,4'-C-ethylene-bridged nucleic acid (ENA)-modified AONs appeared not effective, probably due to lack of nuclear internalization. Selectivity for products from the expanded DMPK allele in patient myoblasts, an important requirement to minimize unwanted side effects, appeared also dependent on AON chemistry. In particular, RNase-H-dependent (CAG)n AONs did not show (CUG)n length specificity. We provide evidence that degradation of long DMPK transcripts induced by PS58-type AONs is an RNase-H independent process, does not involve oligo-intrinsic RNase activity nor does it interfere with splicing of DMPK transcripts. Our collection of triplet repeat AONs forms an important resource for further development of a safe therapy for DM1 and other unstable microsatellite diseases.Molecular Therapy-Nucleic Acids (2013) 2, e81; doi:10.1038/mtna.2013.9; published online 19 March 2013.