Mice lacking MBNL1 and MBNL2 exhibit sudden cardiac death and molecular signatures recapitulating myotonic dystrophy.

Myotonic dystrophy (DM) is caused by expansions of C(C)TG repeats in the non-coding regions of the DMPK and CNBP genes, and DM patients often suffer from sudden cardiac death due to lethal conduction block or arrhythmia. Specific molecular changes that underlie DM cardiac pathology have been linked to repeat-associated depletion of Muscleblind-like (MBNL) 1 and 2 proteins and upregulation of CUGBP, Elav-like family member 1 (CELF1). Hypothesis solely targeting MBNL1 or CELF1 pathways that could address all the consequences of repeat expansion in heart remained inconclusive, particularly when the direct cause of mortality and results of transcriptome analyses remained undetermined in Mbnl compound knockout (KO) mice with cardiac phenotypes. Here, we develop Myh6-Cre double KO (DKO) (Mbnl1-/-; Mbnl2cond/cond; Myh6-Cre+/-) mice to eliminate Mbnl1/2 in cardiomyocytes and observe spontaneous lethal cardiac events under no anesthesia. RNA sequencing recapitulates DM heart spliceopathy and shows gene expression changes that were previously undescribed in DM heart studies. Notably, immunoblotting reveals a nearly 6-fold increase of Calsequestrin 1 and 50% reduction of epidermal growth factor proteins. Our findings demonstrate that complete ablation of MBNL1/2 in cardiomyocytes is essential for generating sudden death due to lethal cardiac rhythms and reveal potential mechanisms for DM heart pathogenesis.

Repeat length increases disease penetrance and severity in C9orf72 ALS/FTD BAC transgenic mice.

C9orf72 ALS/FTD patients show remarkable clinical heterogeneity, but the complex biology of the repeat expansion mutation has limited our understanding of the disease. BAC transgenic mice were used to better understand the molecular mechanisms and repeat length effects of C9orf72 ALS/FTD. Genetic analyses of these mice demonstrate that the BAC transgene and not integration site effects cause ALS/FTD phenotypes. Transcriptomic changes in cell proliferation, inflammation and neuronal pathways are found late in disease and alternative splicing changes provide early molecular markers that worsen with disease progression. Isogenic sublines of mice with 800, 500 or 50 G4C2 repeats generated from the single-copy C9-500 line show longer repeats result in earlier onset, increased disease penetrance and increased levels of RNA foci and dipeptide RAN protein aggregates. These data demonstrate G4C2 repeat length is an important driver of disease and identify alternative splicing changes as early biomarkers of C9orf72 ALS/FTD.

Precise small-molecule cleavage of an r(CUG) repeat expansion in a myotonic dystrophy mouse model.

Myotonic dystrophy type 1 (DM1) is an incurable neuromuscular disorder caused by an expanded CTG repeat that is transcribed into r(CUG)exp The RNA repeat expansion sequesters regulatory proteins such as Muscleblind-like protein 1 (MBNL1), which causes pre-mRNA splicing defects. The disease-causing r(CUG)exp has been targeted by antisense oligonucleotides, CRISPR-based approaches, and RNA-targeting small molecules. Herein, we describe a designer small molecule, Cugamycin, that recognizes the structure of r(CUG)exp and cleaves it in both DM1 patient-derived myotubes and a DM1 mouse model, leaving short repeats of r(CUG) untouched. In contrast, oligonucleotides that recognize r(CUG) sequence rather than structure cleave both long and short r(CUG)-containing transcripts. Transcriptomic, histological, and phenotypic studies demonstrate that Cugamycin broadly and specifically relieves DM1-associated defects in vivo without detectable off-targets. Thus, small molecules that bind and cleave RNA have utility as lead chemical probes and medicines and can selectively target disease-causing RNA structures to broadly improve defects in preclinical animal models.

Transcriptome alterations in myotonic dystrophy skeletal muscle and heart.

Myotonic dystrophy (dystrophia myotonica, DM) is a multi-systemic disease caused by expanded CTG or CCTG microsatellite repeats. Characterized by symptoms in muscle, heart and central nervous system, among others, it is one of the most variable diseases known. A major pathogenic event in DM is the sequestration of muscleblind-like proteins by CUG or CCUG repeat-containing RNAs transcribed from expanded repeats, and differences in the extent of MBNL sequestration dependent on repeat length and expression level may account for some portion of the variability. However, many other cellular pathways are reported to be perturbed in DM, and the severity of specific disease symptoms varies among individuals. To help understand this variability and facilitate research into DM, we generated 120 RNASeq transcriptomes from skeletal and heart muscle derived from healthy and DM1 biopsies and autopsies. A limited number of DM2 and Duchenne muscular dystrophy samples were also sequenced. We analyzed splicing and gene expression, identified tissue-specific changes in RNA processing and uncovered transcriptome changes strongly correlating with muscle strength. We created a web resource at http://DMseq.org that hosts raw and processed transcriptome data and provides a lightweight, responsive interface that enables browsing of processed data across the genome.

Transcriptome-Wide Studies of RNA-Targeted Small Molecules Provide a Simple and Selective r(CUG)exp Degrader in Myotonic Dystrophy.

Myotonic dystrophy type 1 (DM1) is caused by a highly structured RNA repeat expansion, r(CUG)exp, harbored in the 3' untranslated region (3' UTR) of dystrophia myotonica protein kinase (DMPK) mRNA and drives disease through a gain-of-function mechanism. A panel of low-molecular-weight fragments capable of reacting with RNA upon UV irradiation was studied for cross-linking to r(CUG)expin vitro, affording perimidin-2-amine diazirine (1) that bound to r(CUG)exp. The interactions between the small molecule and RNA were further studied by nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. Binding of 1 in DM1 myotubes was profiled transcriptome-wide, identifying 12 transcripts including DMPK that were bound by 1. Augmenting the functionality of 1 with cleaving capability created a chimeric degrader that specifically targets r(CUG)exp for elimination. The degrader broadly improved DM1-associated defects as assessed by RNA-seq, while having limited effects on healthy myotubes. This study (i) provides a platform to investigate molecular recognition of ligands directly in disease-affected cells; (ii) illustrates that RNA degraders can be more specific than the binders from which they are derived; and (iii) suggests that repeating transcripts can be selectively degraded due to the presence of multiple ligand binding sites.

The skeletal muscle circadian clock regulates titin splicing through RBM20.

Circadian rhythms are maintained by a cell-autonomous, transcriptional-translational feedback loop known as the molecular clock. While previous research suggests a role of the molecular clock in regulating skeletal muscle structure and function, no mechanisms have connected the molecular clock to sarcomere filaments. Utilizing inducible, skeletal muscle specific, Bmal1 knockout (iMSBmal1-/-) mice, we showed that knocking out skeletal muscle clock function alters titin isoform expression using RNAseq, liquid chromatography-mass spectrometry, and sodium dodecyl sulfate-vertical agarose gel electrophoresis. This alteration in titin's spring length resulted in sarcomere length heterogeneity. We demonstrate the direct link between altered titin splicing and sarcomere length in vitro using U7 snRNPs that truncate the region of titin altered in iMSBmal1-/- muscle. We identified a mechanism whereby the skeletal muscle clock regulates titin isoform expression through transcriptional regulation of Rbm20, a potent splicing regulator of titin. Lastly, we used an environmental model of circadian rhythm disruption and identified significant downregulation of Rbm20 expression. Our findings demonstrate the importance of the skeletal muscle circadian clock in maintaining titin isoform through regulation of RBM20 expression. Because circadian rhythm disruption is a feature of many chronic diseases, our results highlight a novel pathway that could be targeted to maintain skeletal muscle structure and function in a range of pathologies.

High-content image-based analysis and proteomic profiling identifies Tau phosphorylation inhibitors in a human iPSC-derived glutamatergic neuronal model of tauopathy.

Mutations in MAPT (microtubule-associated protein tau) cause frontotemporal dementia (FTD). MAPT mutations are associated with abnormal tau phosphorylation levels and accumulation of misfolded tau protein that can propagate between neurons ultimately leading to cell death (tauopathy). Recently, a p.A152T tau variant was identified as a risk factor for FTD, Alzheimer's disease, and synucleinopathies. Here we used induced pluripotent stem cells (iPSC) from a patient carrying this p.A152T variant to create a robust, functional cellular assay system for probing pathophysiological tau accumulation and phosphorylation. Using stably transduced iPSC-derived neural progenitor cells engineered to enable inducible expression of the pro-neural transcription factor Neurogenin 2 (Ngn2), we generated disease-relevant, cortical-like glutamatergic neurons in a scalable, high-throughput screening compatible format. Utilizing automated confocal microscopy, and an advanced image-processing pipeline optimized for analysis of morphologically complex human neuronal cultures, we report quantitative, subcellular localization-specific effects of multiple kinase inhibitors on tau, including ones under clinical investigation not previously reported to affect tau phosphorylation. These results demonstrate the potential for using patient iPSC-derived ex vivo models of tauopathy as genetically accurate, disease-relevant systems to probe tau biochemistry and support the discovery of novel therapeutics for tauopathies.

Ribonuclease recruitment using a small molecule reduced c9ALS/FTD r(G4C2) repeat expansion in vitro and in vivo ALS models.

The most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD) is an expanded G4C2 RNA repeat [r(G4C2)exp] in chromosome 9 open reading frame 72 (C9orf72), which elicits pathology through several mechanisms. Here, we developed and characterized a small molecule for targeted degradation of r(G4C2)exp. The compound was able to selectively bind r(G4C2)exp's structure and to assemble an endogenous nuclease onto the target, provoking removal of the transcript by native RNA quality control mechanisms. In c9ALS patient­derived spinal neurons, the compound selectively degraded the mutant C9orf72 allele with limited off-targets and reduced quantities of toxic dipeptide repeat proteins (DPRs) translated from r(G4C2)exp. In vivo work in a rodent model showed that abundance of both the mutant allele harboring the repeat expansion and DPRs were selectively reduced by this compound. These results demonstrate that targeted small-molecule degradation of r(G4C2)exp is a strategy for mitigating c9ALS/FTD-associated pathologies and studying disease-associated pathways in preclinical models.