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

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

Combination of Omics Approaches to Study Molecular Abnormalities in Individual Brain Cell Types of a DM1 Mouse Model

Introduction: Molecular mechanisms of DM1 muscle/cardiac pathology are better characterized than those implicated in CNS dysfunction. To fill this gap, we used the DMSXL mouse model to study different brain cells by multiple omics approaches.Methods: Cells were isolated from cortex of DMSXL/WT littermates, grown and differentiated to obtain homogenous cultures of primary neurons, astrocytes, oligodendrocyte precursor cells and mature oligodendrocytes. RNA samples were analyzed by RNA-Seq to characterize changes in alternative splicing and total gene expression. Total protein and phosphorylation levels were determined by mass spectrometry.Results:RNA-Seq analysis identified new splicing abnormalities in DMSXL brain cells. Gene expression was computed together with (phospho-)proteomics to gain an understanding of the pathways affected in each cell type. Conclusion: Our analysis reveals the importance of combining multiple omics approaches in different CNS cells to shed new light on the pathways affected and provide an integrative view of DM1 brain dysfunction

Toxic RNA affects astrocyte adhesion, spreading and migration in myotonic dystrophy, and impacts neuritogenesis through abnormal glial-neuronal interactions

International audienceMyotonic dystrophy type 1 (DM1) is a multisystemic condition that affects many tissues, as well as age groups. Compelling clinical evidence clearly demonstrates the impairment of the central nervous system (CNS), through cognitive/attention deficits, executive dysfunction, prevalent hypersomnia, behavioral changes and intellectual disability in the most severe cases. The neurological manifestations are highly debilitating and distressing for patients and their relatives, and there is no cure for this devastating condition.DM1 is caused by the abnormal expansion of a non-coding trinucleotide CTG repeats. Expanded CUG transcripts accumulate in toxic RNA aggregates or foci in the cell nucleus, perturbing the activity of key RNA-binding proteins and deregulating the splicing and general RNA metabolism of downstream targets. However, important gaps exist in our understanding of the disease mechanisms in the brain. In particular, we do not know the cell types primarily affected or the molecular pathways mostly dysregulated by the repeat expansion in the CNS. Using a transgenic mouse model of DM1 we found preferential accumulation of toxic RNA foci and missplicing in cortical astrocytes relative to neurons, pointing to glia pathology. We then used our DM1 mice as a source of primary neurons and astrocytes to resolve cell type-specific phenotypes and their associated molecular abnormalities. DM1 primary astrocytes show greater RNA foci accumulation and missplicing, relative to neurons, in association with defective cell growth, adhesion, cell spreading, polarization and migration. In contrast, the growth profile of DM1 primary neurons remained unaltered, but late neurite arborization was significantly impaired. Interestingly, defects in neuritogenesis were aggravated by the presence of DM1 mouse astrocytes in co-culture systems. To dissect the molecular mechanisms behind astrocyte dysfunction we performed global proteomics and transcriptomics approaches on homogenous astrocyte cultures. In line with the cell phenotypes described, we found relevant splicing and expression changes in critical regulators of cytoskeletal dynamics and cell adhesion.In conclusion, the DM1 repeat expansion has a deleterious impact on glia cell biology, which may in turn affect neuronal physiology through defective glial-neuronal crosstalk. Our results provide new insight into the cellular and molecular mechanisms of DM1 brain disease

Toxic CUG RNA repeats disrupt developmentally-regulated splicing in oligodendrocytes causing transient hypomyelination in a mouse model of myotonic dystrophy.

International audienceMyotonic dystrophy type 1 (DM1) is a neuromuscular disorder, characterised by cognitive and behavioural impairment, in addition to the typical muscle pathology. Imaging studies revealed widespread white matter lesions in DM1 patients, which may be partially explained by local demyelination. DM1 is caused by the abnormal expansion of non-coding CTG trinucleotide repeat, which is transcribed into toxic CUG RNA that accumulates in nuclear RNA foci and disrupts critical RNA-binding proteins. To investigate the impact of RNA toxicity on myelin biology, we used a transgenic mouse model of DM1, known as the DMSXL mice, which express expanded CUG transcripts in multiple tissues and cell types.We found delayed axon myelination of the corpus callosum at two weeks of age, which later recovered at four months. Early myelin defects were linked to an overall reduction in the number of oligodendroglia cells, specifically myelinating oligodendrocytes (OL), with a concurrent increase in oligodendrocyte progenitor cells (OPC). These phenotypes were accompanied by abundant RNA foci and splicing dysregulation in oligodendroglia, which were more pronounced at two weeks. Taking advantage of primary cell models, we investigated the mechanisms triggered by toxic CUG RNA in oligodendroglia. OPC isolated from DMSXL newborns exhibited impaired differentiation into fully ramified OLs in culture, a defect we confirmed in human oligodendroglia derived from DM1 patients. RNA sequencing revealed expression and splicing changes in DMSXL OL that affect transcripts related to the cytoskeleton and cell differentiation. Importantly, the transcriptomic defects of DMSXL OL recreated expression and splicing profiles typical of immature OPC.In conclusion, toxic CUG RNA disrupts the molecular program of oligodendroglia differentiation, impairing the transcriptome changes occurring during the OPC-OL transition and leading to transient hypomyelination in mice

Toxic CUG RNA repeats disrupt developmentally-regulated splicing in oligodendrocytes causing transient hypomyelination in a mouse model of myotonic dystrophy.

International audienceMyotonic dystrophy type 1 (DM1) is a neuromuscular disorder, characterised by cognitive and behavioural impairment, in addition to the typical muscle pathology. Imaging studies revealed widespread white matter lesions in DM1 patients, which may be partially explained by local demyelination. DM1 is caused by the abnormal expansion of non-coding CTG trinucleotide repeat, which is transcribed into toxic CUG RNA that accumulates in nuclear RNA foci and disrupts critical RNA-binding proteins. To investigate the impact of RNA toxicity on myelin biology, we used a transgenic mouse model of DM1, known as the DMSXL mice, which express expanded CUG transcripts in multiple tissues and cell types.We found delayed axon myelination of the corpus callosum at two weeks of age, which later recovered at four months. Early myelin defects were linked to an overall reduction in the number of oligodendroglia cells, specifically myelinating oligodendrocytes (OL), with a concurrent increase in oligodendrocyte progenitor cells (OPC). These phenotypes were accompanied by abundant RNA foci and splicing dysregulation in oligodendroglia, which were more pronounced at two weeks. Taking advantage of primary cell models, we investigated the mechanisms triggered by toxic CUG RNA in oligodendroglia. OPC isolated from DMSXL newborns exhibited impaired differentiation into fully ramified OLs in culture, a defect we confirmed in human oligodendroglia derived from DM1 patients. RNA sequencing revealed expression and splicing changes in DMSXL OL that affect transcripts related to the cytoskeleton and cell differentiation. Importantly, the transcriptomic defects of DMSXL OL recreated expression and splicing profiles typical of immature OPC.In conclusion, toxic CUG RNA disrupts the molecular program of oligodendroglia differentiation, impairing the transcriptome changes occurring during the OPC-OL transition and leading to transient hypomyelination in mice

MBNL dependent-impaired development connectivity within neuromuscular circuits in Myotonic Dystrophy type.

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MBNL dependent-impaired development connectivity within neuromuscular circuits in Myotonic Dystrophy type.

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RNA toxicity in myotonic dystrophy causes pronounced spliceopathy in astrocytes, in association with defective cell adhesion and morphology, erratic migration and impaired polarization

International audienceMyotonic dystrophy type 1 (DM1) is a severe multisystemic condition. The impairment of the central nervous system (CNS) is demonstrated by cognitive and attention deficits, executive dysfunction, prevalent hypersomnia, behavioral changes, as well as intellectual disability in the most severe cases. DM1 is caused by the abnormal expansion of a non-coding trinucleotide CTG repeat. Expanded CUG transcripts accumulate in toxic RNA aggregates (or foci) in the cell nucleus, which perturb primarily the regulation of alternative splicing. Important gaps still exist in our understanding of the disease mechanisms in the brain: we do not know the cell types and the molecular pathways most predominantly affected, and how they contribute to the onset of the debilitating neurological manifestations of DM1.Using a transgenic mouse model of DM1 we found preferential accumulation of toxic RNA foci and missplicing in cortical astrocytes, relative to neurons, pointing to glia cell pathology. We used our DM1 mice as a source of primary neurons and astrocytes to resolve cell type-specific disease mechanisms by RNA sequencing of homogenous cell cultures. DM1 mouse astrocytes confirmed greater RNA foci accumulation and showed critical missplicing of transcripts that regulate cell adhesion, cytoskeleton dynamics and cell morphogenesis. Astrocyte spliceopathy translated into defective cell adhesion, reduced spreading and erratic migration in culture, as well as decreased astrocyte ramification and aberrant reorientation in DM1 mouse brains. We confirmed the abnormal splicing of relevant transcripts in brain tissue from DM1 patients, and the defective spreading of human glia cells expressing toxic CUG RNA in culture.In conclusion, we have shown the CTG repeat expansion has a deleterious impact on glia cell biology, which may impair the glial-neuronal crosstalk and synaptic function in DM1 brains, contributing to cognitive and behavioural deficits. Our results provide new insight into the cellular and molecular mechanisms of DM1 brain disease