Combinatorial effects of ion channel mis-splicing as a cause of myopathy in myotonic dystrophy.

Myotonic dystrophy type 1 (DM1) is an autosomal dominant disorder caused by an unstable expanded CTG repeat located in the 3'-UTR of the DM1 protein kinase (DMPK) gene. The pathogenic mechanism results in misregulated alternative splicing of hundreds of genes, creating the dilemma of establishing which genes contribute to the mechanism of DM1 skeletal muscle pathology. In this issue of the JCI, Cisco and colleagues systematically tested the combinatorial effects of DM1-relevant mis-splicing patterns in vivo and identified the synergistic effects of mis-spliced calcium and chloride channels as a major contributor to DM1 skeletal muscle impairment. The authors further demonstrated the therapeutic potential for calcium channel modulation to block the synergistic effects and rescue myopathy.

The role of Limch1 alternative splicing in skeletal muscle function.

Postnatal skeletal muscle development is a highly dynamic period associated with widespread alternative splicing changes required to adapt tissues to adult function. These splicing events have significant implications because the reversion of adult mRNA isoforms to fetal isoforms is observed in forms of muscular dystrophy. LIMCH1 is a stress fiber-associated protein that is alternatively spliced to generate uLIMCH1, a ubiquitously expressed isoform, and mLIMCH1, a skeletal muscle-specific isoform containing six additional exons simultaneously included after birth in the mouse. CRISPR/Cas9 was used to delete the six alternatively spliced exons of LIMCH1 in mice, thereby forcing the constitutive expression of the predominantly fetal isoform, uLIMCH1. mLIMCH1 knockout mice had significant grip strength weakness in vivo, and maximum force generated was decreased ex vivo. Calcium-handling deficits were observed during myofiber stimulation that could explain the mechanism by which mLIMCH1 knockout leads to muscle weakness. In addition, LIMCH1 is mis-spliced in myotonic dystrophy type 1, with the muscleblind-like (MBNL) family of proteins acting as the likely major regulator of Limch1 alternative splicing in skeletal muscle.

Insights into Cell-Specific Functions of Microtubules in Skeletal Muscle Development and Homeostasis.

The contractile cells of skeletal muscles, called myofibers, are elongated multinucleated syncytia formed and maintained by the fusion of proliferative myoblasts. Human myofibers can be hundreds of microns in diameter and millimeters in length. Myofibers are non-mitotic, obviating the need for microtubules in cell division. However, microtubules have been adapted to the unique needs of these cells and are critical for myofiber development and function. Microtubules in mature myofibers are highly dynamic, and studies in several experimental systems have demonstrated the requirements for microtubules in the unique features of muscle biology including myoblast fusion, peripheral localization of nuclei, assembly of the sarcomere, transport and signaling. Microtubule-binding proteins have also been adapted to the needs of the skeletal muscle including the expression of skeletal muscle-specific protein isoforms generated by alternative splicing. Here, we will outline the different roles microtubules play in skeletal muscle cells, describe how microtubule abnormalities can lead to muscle disease and discuss the broader implications for microtubule function.

Alternative splicing mediates the compensatory upregulation of MBNL2 upon MBNL1 loss-of-function.

Loss of gene function can be compensated by paralogs with redundant functions. An example of such compensation are the paralogs of the Muscleblind-Like (MBNL) family of RNA-binding proteins that are sequestered and lose their function in Myotonic Dystrophy Type 1 (DM1). Loss of MBNL1 increases the levels of its paralog MBNL2 in tissues where Mbnl2 expression is low, allowing MBNL2 to functionally compensate for MBNL1 loss. Here, we show that loss of MBNL1 increases the inclusion of Mbnl2 exon 6 and exon 9. We find that inclusion of Mbnl2 exon 6 increases the translocation of MBNL2 to the nucleus, while the inclusion of Mbnl2 exon 9 shifts the reading frame to an alternative C-terminus. We show that the C-terminus lacking exon 9 contains a PEST domain which causes proteasomal degradation. Loss of MBNL1 increases the inclusion of exon 9, resulting in an alternative C-terminus lacking the PEST domain and the increase of MBNL2. We further find that the compensatory mechanism is active in a mouse DM1 model. Together, this study uncovers the compensatory mechanism by which loss of MBNL1 upregulates its paralog MBNL2 and highlights a potential role of the compensatory mechanism in DM1.

Toward a best-form CPC-like nonimaging optical concentrator.

We present near-ideal axisymmetric numerically optimized spline concentrators (OSCs) which outperform the compound parabolic concentrator (CPC). By perturbing the profile of the revolved CPC by a variable-offset spline defined in tangent-normal space, we show that ray rejection can be reduced to nearly half of that of the CPC, without increasing concentrator length. The resulting OSCs achieve acceptance efficiencies as high as 99.3% for an acceptance angle of 45°, the highest reported for any finite-length CPC-like light concentrator. A set of design curves is presented which can be used to generate near "best-form" OSCs for any acceptance angle in the range 10° to 45°.

Clinical and Molecular Insights into Gastrointestinal Dysfunction in Myotonic Dystrophy Types 1 & 2.

Myotonic dystrophy (DM) is a highly variable, multisystemic disorder that clinically affects one in 8000 individuals. While research has predominantly focused on the symptoms and pathological mechanisms affecting striated muscle and brain, DM patient surveys have identified a high prevalence for gastrointestinal (GI) symptoms amongst affected individuals. Clinical studies have identified chronic and progressive dysfunction of the esophagus, stomach, liver and gallbladder, small and large intestine, and rectum and anal sphincters. Despite the high incidence of GI dysmotility in DM, little is known regarding the pathological mechanisms leading to GI dysfunction. In this review, we summarize results from clinical and molecular analyses of GI dysfunction in both genetic forms of DM, DM type 1 (DM1) and DM type 2 (DM2). Based on current knowledge of DM primary pathological mechanisms in other affected tissues and GI tissue studies, we suggest that misregulation of alternative splicing in smooth muscle resulting from the dysregulation of RNA binding proteins muscleblind-like and CUGBP-elav-like is likely to contribute to GI dysfunction in DM. We propose that a combinatorial approach using clinical and molecular analysis of DM GI tissues and model organisms that recapitulate DM GI manifestations will provide important insight into defects impacting DM GI motility.

Reversible cardiac disease features in an inducible CUG repeat RNA-expressing mouse model of myotonic dystrophy.

Myotonic dystrophy type 1 (DM1) is caused by a CTG repeat expansion in the DMPK gene. Expression of pathogenic expanded CUG repeat (CUGexp) RNA causes multisystemic disease by perturbing the functions of RNA-binding proteins, resulting in expression of fetal protein isoforms in adult tissues. Cardiac involvement affects 50% of individuals with DM1 and causes 25% of disease-related deaths. We developed a transgenic mouse model for tetracycline-inducible and heart-specific expression of human DMPK mRNA containing 960 CUG repeats. CUGexp RNA is expressed in atria and ventricles and induced mice exhibit electrophysiological and molecular features of DM1 disease, including cardiac conduction delays, supraventricular arrhythmias, nuclear RNA foci with Muscleblind protein colocalization, and alternative splicing defects. Importantly, these phenotypes were rescued upon loss of CUGexp RNA expression. Transcriptome analysis revealed gene expression and alternative splicing changes in ion transport genes that are associated with inherited cardiac conduction diseases, including a subset of genes involved in calcium handling. Consistent with RNA-Seq results, calcium-handling defects were identified in atrial cardiomyocytes isolated from mice expressing CUGexp RNA. These results identify potential tissue-specific mechanisms contributing to cardiac pathogenesis in DM1 and demonstrate the utility of reversible phenotypes in our model to facilitate development of targeted therapeutic approaches.

Overlapping mechanisms of lncRNA and expanded microsatellite RNA.

RNA has major regulatory roles in a wide range of biological processes and a surge of RNA research has led to the classification of numerous functional RNA species. One example is long noncoding RNAs (lncRNAs) that are structurally complex transcripts >200 nucleotides (nt) in length and lacking a canonical open reading frame (ORF). Despite a general lack of sequence conservation and low expression levels, many lncRNAs have been shown to have functionality in diverse biological processes as well as in mechanisms of disease. In parallel with the growing understanding of lncRNA functions, there is a growing subset of microsatellite expansion disorders in which the primary mechanism of pathogenesis is an RNA gain of function arising from RNA transcripts from the mutant allele. Microsatellite expansion disorders are caused by an expansion of short (3-10 nt) repeats located within coding genes. Expanded repeat-containing RNA mediates toxicity through multiple mechanisms, the details of which remain only partially understood. The purpose of this review is to highlight the links between functional mechanisms of lncRNAs and the potential pathogenic mechanisms of expanded microsatellite RNA. These shared mechanisms include protein sequestration, peptide translation, micro-RNA (miRNA) processing, and miRNA sequestration. Recognizing the parallels between the normal functions of lncRNAs and the negative impact of expanded microsatellite RNA on biological processes can provide reciprocal understanding to the roles of both RNA species. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications RNA in Disease and Development > RNA in Disease.

Increased nuclear but not cytoplasmic activities of CELF1 protein leads to muscle wasting.

mRNA processing is highly regulated during development through changes in RNA-binding protein (RBP) activities. CUG-BP, Elav-like family member 1 (CELF1, also called CUGBP1) is an RBP, the expression of which decreases in skeletal muscle soon after birth. CELF1 regulates multiple nuclear and cytoplasmic RNA processing events. In the nucleus, CELF1 regulates networks of postnatal alternative splicing (AS) transitions, while in the cytoplasm, CELF1 regulates mRNA stability and translation. Stabilization and misregulation of CELF1 has been implicated in human diseases including myotonic dystrophy type 1, Alzheimer's disease and multiple cancers. To understand the contribution of nuclear and cytoplasmic CELF1 activity to normal and pathogenic skeletal muscle biology, we generated transgenic mice for doxycycline-inducible and skeletal muscle-specific expression of active CELF1 mutants engineered to be localized predominantly to either the nucleus or the cytoplasm. Adult mice expressing nuclear, but not cytoplasmic, CELF1 are characterized by strong histopathological defects, muscle loss within 10 days and changes in AS. In contrast, mice expressing cytoplasmic CELF1 display changes in protein levels of targets known to be regulated at the level of translation by CELF1, with minimal changes in AS. These changes are in the absence of overt histopathological changes or muscle loss. RNA-sequencing revealed extensive gene expression and AS changes in mice overexpressing nuclear and naturally localized CELF1 protein, with affected genes involved in cytoskeleton dynamics, membrane dynamics, RNA processing and zinc ion binding. These results support a stronger role for nuclear CELF1 functions as compared to cytoplasmic CELF1 functions in skeletal muscle wasting.

Aberrant Expression of a Non-muscle RBFOX2 Isoform Triggers Cardiac Conduction Defects in Myotonic Dystrophy.

Myotonic dystrophy type 1 (DM1) is a multisystemic genetic disorder caused by the CTG repeat expansion in the 3'-untranslated region of DMPK gene. Heart dysfunctions occur in ∼80% of DM1 patients and are the second leading cause of DM1-related deaths. Herein, we report that upregulation of a non-muscle splice isoform of RNA-binding protein RBFOX2 in DM1 heart tissue-due to altered splicing factor and microRNA activities-induces cardiac conduction defects in DM1 individuals. Mice engineered to express the non-muscle RBFOX240 isoform in heart via tetracycline-inducible transgenesis, or CRISPR/Cas9-mediated genome editing, reproduced DM1-related cardiac conduction delay and spontaneous episodes of arrhythmia. Further, by integrating RNA binding with cardiac transcriptome datasets from DM1 patients and mice expressing the non-muscle RBFOX2 isoform, we identified RBFOX240-driven splicing defects in voltage-gated sodium and potassium channels, which alter their electrophysiological properties. Thus, our results uncover a trans-dominant role for an aberrantly expressed RBFOX240 isoform in DM1 cardiac pathogenesis.

Endurance exercise leads to beneficial molecular and physiological effects in a mouse model of myotonic dystrophy type 1.

INTRODUCTION: Myotonic dystrophy type 1 (DM1) is a multisystemic disease caused by expansion of a CTG repeat in the 3' UTR of the Dystrophia Myotonica-Protein Kinase (DMPK) gene. While multiple organs are affected, more than half of mortality is due to muscle wasting. METHODS: It is unclear whether endurance exercise provides beneficial effects in DM1. Here, we show that a 10-week treadmill endurance exercise program leads to beneficial effects in the HSALR mouse model of DM1. RESULTS: Animals that performed treadmill training displayed reduced CUGexp RNA levels, improved splicing abnormalities, an increase in skeletal muscle weight and improved endurance capacity. DISCUSSION: These results indicate that endurance exercise does not have adverse effects in HSALR animals and contributes to beneficial molecular and physiological outcomes.

Harnessing Heat Beyond 200 °C from Unconcentrated Sunlight with Nonevacuated Transparent Aerogels.

Heat at intermediate temperatures (120-220 °C) is in significant demand in both industrial and domestic sectors for applications such as water and space heating, steam generation, sterilization, and other industrial processes. Harnessing heat from solar energy at these temperatures, however, requires costly optical and mechanical components to concentrate the dilute solar flux and suppress heat losses. Thus, achieving high temperatures under unconcentrated sunlight remains a technological challenge as well as an opportunity for utilizing solar thermal energy. In this work, we demonstrate a solar receiver capable of reaching over 265 °C under ambient conditions without optical concentration. The high temperatures are achieved by leveraging an artificial greenhouse effect within an optimized monolithic silica aerogel to reduce heat losses while maintaining high solar transparency. This study demonstrates a viable path to promote cost-effective solar thermal energy at intermediate temperatures.

Alu insertion variants alter mRNA splicing.

RNA splicing is a highly regulated process dependent on sequences near splice sites. Insertions of Alu retrotransposons can disrupt splice sites or bind splicing regulators. We hypothesized that some common inherited polymorphic Alu insertions are responsible for splicing QTLs (sQTL). We focused on intronic Alu variants mapping within 100 bp of an alternatively used exon and screened for those that alter splicing. We identify five loci, 21.7% of those assayed, where the polymorphic Alu alters splicing. While in most cases the Alu promotes exon skipping, at one locus the Alu increases exon inclusion. Of particular interest is an Alu polymorphism in the CD58 gene. Reduced CD58 expression is associated with risk for developing multiple sclerosis. We show that the Alu insertion promotes skipping of CD58 exon 3 and results in a frameshifted transcript, indicating that the Alu may be the causative variant for increased MS risk at this locus. Using RT-PCR analysis at the endogenous locus, we confirm that the Alu variant is a sQTL for CD58. In summary, altered splicing efficiency is a common functional consequence of Alu polymorphisms including at least one instance where the variant is implicated in disease risk. This work broadens our understanding of splicing regulatory sequences around exons.

Contactless steam generation and superheating under one sun illumination.

Steam generation using solar energy provides the basis for many sustainable desalination, sanitization, and process heating technologies. Recently, interest has arisen for low-cost floating structures that absorb solar radiation and transfer energy to water via thermal conduction, driving evaporation. However, contact between water and the structure leads to fouling and pins the vapour temperature near the boiling point. Here we demonstrate solar-driven evaporation using a structure not in contact with water. The structure absorbs solar radiation and re-radiates infrared photons, which are directly absorbed by the water within a sub-100 µm penetration depth. Due to the physical separation from the water, fouling is entirely avoided. Due to the thermal separation, the structure is no longer pinned at the boiling point, and is used to superheat the generated steam. We generate steam with temperatures up to 133 °C, demonstrating superheated steam in a non-pressurized system under one sun illumination.

CRISPR -Mediated Expression of the Fetal Scn5a Isoform in Adult Mice Causes Conduction Defects and Arrhythmias.

Background The sodium channel, Nav1.5, encoded by SCN 5A, undergoes developmentally regulated splicing from inclusion of exon 6A in the fetal heart to exon 6B in adults. These mutually exclusive exons differ in 7 amino acids altering the electrophysiological properties of the Nav1.5 channel. In myotonic dystrophy type 1, SCN 5A is mis-spliced such that the fetal pattern of exon 6A inclusion is detected in adult hearts. Cardiac manifestations of myotonic dystrophy type 1 include conduction defects and arrhythmias and are the second-leading cause of death. Methods and Results This work aimed to determine the impact of SCN 5A mis-splicing on cardiac function. We used clustered regularly interspaced short palindromic repeat ( CRISPR) /CRISPR-associated protein 9 (Cas9) to delete Scn5a exon 6B in mice, thereby redirecting splicing toward exon 6A. These mice exhibit prolonged PR and QRS intervals, slowed conduction velocity, extended action potential duration, and are highly susceptible to arrhythmias. Conclusions Our findings highlight a nonmutational pathological mechanism of arrhythmias and conduction defects as a result of mis-splicing of the predominant cardiac sodium channel. Animals homozygous for the deleted exon express only the fetal isoform and have more-severe phenotypes than heterozygotes that also express the adult isoform. This observation is directly relevant to myotonic dystrophy type 1, and possibly pathological arrhythmias, in which individuals differ with regard to the ratios of the isoforms expressed.

Modulation of alternative splicing of trafficking genes by genome editing reveals functional consequences in muscle biology.

Alternative splicing is a regulatory mechanism by which multiple mRNA isoforms are generated from single genes. Numerous genes that encode membrane trafficking proteins are alternatively spliced. However, there is limited information about the functional consequences that result from these splicing transitions. Here, we developed appropriate tools to study the functional impact of alternative splicing in development within the most in vivo context. Secondly, we provided evidence of the physiological implications of splicing regulation during muscle development. Our previous work in mouse heart development identified three trafficking genes that are regulated by alternative splicing between birth and adulthood: the clathrin heavy chain, the clathrin light chain-a, and the trafficking kinesin binding protein-1. Here, we demonstrated that alternative splicing regulation of these three genes is tissue- and developmental stage-specific. To identify the functional consequences of splicing regulation in vivo, we used genome editing to block the neonatal-to-adult splicing transitions. We characterized the phenotype of one of these mouse lines and demonstrated that when splicing regulation of the clathrin heavy chain gene is prevented mice exhibit an increase in body and muscle weights which is due to an enlargement in myofiber size. The significance of this work has two components. First, we revealed novel roles of the clathrin heavy chain in muscle growth and showed that its regulation by alternative splicing contributes to muscle development. Second, the new mouse lines will provide a useful tool to study how splicing regulation of three trafficking genes affects tissue identity acquisition and maturation in vivo.

Rbfox-Splicing Factors Maintain Skeletal Muscle Mass by Regulating Calpain3 and Proteostasis.

Maintenance of skeletal muscle mass requires a dynamic balance between protein synthesis and tightly controlled protein degradation by the calpain, autophagy-lysosome, and ubiquitin-proteasome systems (proteostasis). Several sensing and gene-regulatory mechanisms act together to maintain this balance in response to changing conditions. Here, we show that deletion of the highly conserved Rbfox1 and Rbfox2 alternative splicing regulators in adult mouse skeletal muscle causes rapid, severe loss of muscle mass. Rbfox deletion did not cause a reduction in global protein synthesis, but it led to altered splicing of hundreds of gene transcripts, including capn3, which produced an active form of calpain3 protease. Rbfox knockout also led to a reduction in autophagy flux, likely producing a compensatory increase in general protein degradation by the proteasome. Our results indicate that the Rbfox-splicing factors are essential for the maintenance of skeletal muscle mass and proteostasis.

Mechanisms of skeletal muscle wasting in a mouse model for myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is a multi-systemic disease resulting in severe muscle weakening and wasting. DM1 is caused by expansion of CTG repeats in the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. We have developed an inducible, skeletal muscle-specific mouse model of DM1 (CUG960) that expresses 960 CUG repeat-expressing animals (CUG960) in the context of human DMPK exons 11-15. CUG960 RNA-expressing mice induced at postnatal day 1, as well as adult-onset animals, show clear, measurable muscle wasting accompanied by severe histological defects including central myonuclei, reduced fiber cross-sectional area, increased percentage of oxidative myofibers, the presence of nuclear RNA foci that colocalize with Mbnl1 protein, and increased Celf1 protein in severely affected muscles. Importantly, muscle loss, histological abnormalities and RNA foci are reversible, demonstrating recovery upon removal of toxic RNA. RNA-seq and protein array analysis indicate that the balance between anabolic and catabolic pathways that normally regulate muscle mass may be disrupted by deregulation of platelet derived growth factor receptor ß signaling and the PI3K/AKT pathways, along with prolonged activation of AMP-activated protein kinase α signaling. Similar changes were detected in DM1 skeletal muscle compared with unaffected controls. The mouse model presented in this paper shows progressive skeletal muscle wasting and has been used to identify potential molecular mechanisms underlying skeletal muscle loss. The reversibility of the phenotype establishes a baseline response for testing therapeutic approaches.

Correction: Impaired Mitochondrial Energy Production Causes Light-Induced Photoreceptor Degeneration Independent of Oxidative Stress.

[This corrects the article DOI: 10.1371/journal.pbio.1002197.].

A Therapeutic Double Whammy: Transcriptional or Post-transcriptional Suppression of Microsatellite Repeat Toxicity by Cas9.

Microsatellite expansion diseases are caused by unstable tandem repeats of 3-10 nucleotides that become pathogenic beyond a threshold number of copies. Two groups present different approaches to reduce pathogenesis by targeting deactivated Cas9 to either the DNA (Pinto et al., 2017) or the RNA (Batra et al., 2017) repeats with therapeutic potential for several diseases.