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Myotonic Dystrophy type 1 (DM1), a highly prevalent form of muscular dystrophy, is caused by (CTG) n repeat expansion in the DMPK gene. Much of DM1 research has focused on the effects within the muscle and neurological tissues; however, DM1 patients also suffer from various metabolic and liver dysfunctions such as increased susceptibility to metabolic dysfunction-associated fatty liver disease (MAFLD) and heightened sensitivity to certain drugs. Here, we generated a liver-specific DM1 mouse model that reproduces molecular and pathological features of the disease, including susceptibility to MAFLD and reduced capacity to metabolize specific analgesics and muscle relaxants. Expression of CUG-expanded (CUG) exp repeat RNA within hepatocytes sequestered muscleblind-like proteins and triggered widespread gene expression and RNA processing defects. Mechanistically, we demonstrate that increased expression and alternative splicing of acetyl-CoA carboxylase 1 drives excessive lipid accumulation in DM1 livers, which is exacerbated by high-fat, high-sugar diets. Together, these findings reveal that (CUG) exp RNA toxicity disrupts normal hepatic functions, predisposing DM1 livers to injury, MAFLD, and drug clearance pathologies that may jeopardize the health of affected individuals and complicate their treatment., (© 2024. The Author(s).)

Myotonic Dystrophy Type 1 (DM1) is an autosomal dominant multisystemic disorder for which cardiac features, including conduction delays and arrhythmias, are the second leading cause of disease mortality. DM1 is caused by expanded CTG repeats in the 3' untranslated region of the DMPK gene. Transcription of the expanded DMPK allele produces mRNAs containing long tracts of CUG repeats, which sequester the Muscleblind-Like family of RNA binding proteins, leading to their loss-of-function and the dysregulation of alternative splicing. A well-characterized mis-regulated splicing event in the DM1 heart is the increased inclusion of SCN5A exon 6A rather than the mutually exclusive exon 6B that normally predominates in adult heart. As previous work showed that forced inclusion of Scn5a exon 6A in mice recapitulates cardiac DM1 phenotypes, we tested whether rescue of Scn5a mis-splicing would improve the cardiac phenotypes in a DM1 heart mouse model. We generated mice lacking Scn5a exon 6A to force the expression of the adult SCN5A isoform including exon 6B and crossed these mice to our previously established CUG960 DM1 heart mouse model. We showed that correction Scn5a mis-splicing does not improve the DM1 heart conduction delays and structural changes induced by CUG repeat RNA expression. Interestingly, we found that in addition to Scn5a mis-splicing, Scn5a expression is reduced in heart tissues of CUG960 mice and DM1-affected individuals. These data indicate that Scn5a mis-splicing is not the sole driver of DM1 heart deficits and suggest a potential role for reduced Scn5a expression in DM1 cardiac disease., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Cell-based strategies are being explored as a therapeutic option for muscular dystrophies, using a variety of cell types from different origin and with different characteristics. Primary pericytes are multifunctional cells found in the capillary bed that exhibit stem cell-like and myogenic regenerative properties. This unique combination allows them to be applied systemically, presenting a promising opportunity for body-wide muscle regeneration. We previously reported the successful isolation of ALP + pericytes from skeletal muscle of patients with myotonic dystrophy type 1 (DM1). These pericytes maintained normal growth parameters and myogenic characteristics in vitro despite the presence of nuclear (CUG) n RNA foci, the cellular hallmark of DM1. Here, we examined the behaviour of DM1 pericytes during myogenic differentiation., Methods: DMPK (CTG) n repeat lengths in patient pericytes were assessed using small pool PCR, to be able to relate variation in myogenic properties and disease hallmarks to repeat expansion. Pericytes from unaffected controls and DM1 patients were cultured under differentiating conditions in vitro. In addition, the pericytes were grown in co-cultures with myoblasts to examine their regenerative capacity by forming hybrid myotubes. Finally, the effect of pericyte fusion on DM1 disease hallmarks was investigated., Results: Small pool PCR analysis revealed the presence of somatic mosaicism in pericyte cell pools. Upon differentiation to myotubes, DMPK expression was upregulated, leading to an increase in nuclear foci sequestering MBNL1 protein. Remarkably, despite the manifestation of these disease biomarkers, patient-derived pericytes demonstrated myogenic potential in co-culture experiments comparable to unaffected pericytes and myoblasts. However, only the unaffected pericytes improved the disease hallmarks in hybrid myotubes. From 20% onwards, the fraction of unaffected nuclei in myotubes positively correlated with a reduction of the number of RNA foci and an increase in the amount of free MBNL1., Conclusions: Fusion of only a limited number of unaffected myogenic precursors to DM1 myotubes already ameliorates cellular disease hallmarks, offering promise for the development of cell transplantation strategies to lower disease burden., (© 2024. The Author(s).)

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.

Myotonic dystrophy type 1 (DM1) is a multi-systemic disorder caused by expansion of CTG microsatellite repeats within DMPK. The most severe form, congenital myotonic dystrophy (CDM), has symptom onset at birth due to large intergenerational repeat expansions. Despite a common mutation, CDM individuals present with a distinct clinical phenotype and absence of common DM1 symptoms. Given the clinical divergence, it is unknown if the hallmark of DM1 pathology, dysregulation of alternative splicing (AS) due to sequestration of MBNL proteins within toxic CUG repeat RNAs, contributes to disease throughout pediatric development. To evaluate global transcriptomic dysregulation, RNA-seq was performed on 36 CDM skeletal muscle biopsies ages 2 weeks to 16 years, including two longitudinal samples. Fifty DM1 and adult/pediatric controls were also sequenced as comparative groups. Despite a large CTG expansion and shared age of onset, CDM individuals presented with a heterogenous, MBNL-dependent mis-splicing signature. Estimation of intracellular MBNL concentrations from splicing responses of select events correlated with total spliceopathy and revealed a distinct, triphasic pattern of AS dysregulation across pediatric development. CDM infants (< 2 years) possess severe mis-splicing that significantly improves in early childhood (2-8 years) independent of sex or CTG repeat load. Adolescent individuals (8-16 years) stratified into two populations with a full range of global splicing dysregulation. DMPK expression changes correlated with alterations in splicing severity during development. This study reveals the complex dynamics of the CDM muscle transcriptome and provides insights into new therapeutic strategies, timing of therapeutic intervention, and biomarker development., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Protein kinases of the dystonia myotonica protein kinase (DMPK) family are critical regulators of actomyosin contractility in cells. The DMPK kinase MRCK1 is required for the activation of myosin, leading to the development of cortical tension, apical constriction, and early gastrulation. Here, we present the structure, conformation, and membrane-binding properties of Caenorhabditis elegans MRCK1. MRCK1 forms a homodimer with N-terminal kinase domains, a parallel coiled coil of 55 nm, and a C-terminal tripartite module of C1, pleckstrin homology (PH), and citron homology (CNH) domains. We report the high-resolution structure of the membrane-binding C1-PH-CNH module of MRCK1 and, using high-throughput and conventional liposome-binding assays, determine its binding to specific phospholipids. We further characterize the interaction of the C-terminal CRIB motif with Cdc42. The length of the coiled-coil domain of DMPK kinases is remarkably conserved over millions of years of evolution, suggesting that they may function as molecular rulers to position kinase activity at a fixed distance from the membrane., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Reduced brain volume including atrophy in grey and white matter is commonly seen in myotonic dystrophy type 1 (DM1). DM1 is caused by an expansion of CTG trinucleotide repeats in the 3' untranslated region (UTR) of the Dystrophia Myotonica Protein Kinase (DMPK) gene. Mutant DMPK mRNA containing expanded CUG RNA (DMPK-CUG exp ) sequesters cytoplasmic MBNL1, resulting in morphological impairment. How DMPK-CUG exp and loss of MBNL1 cause histopathological phenotypes in the DM1 brain remains elusive. Here, we show that BDNF-TrkB retrograde transport is impaired in neurons expressing DMPK-CUG exp due to loss of cytoplasmic MBNL1 function. We reveal that mature BDNF protein levels are reduced in the brain of the DM1 mouse model EpA960/CaMKII-Cre. Exogenous BDNF treatment did not rescue impaired neurite outgrowth in neurons expressing DMPK-CUG exp , whereas overexpression of the cytoplasmic MBNL1 isoform in DMPK-CUG exp -expressing neurons improved their responsiveness to exogenous BDNF. We identify dynein light chain LC8-type 2, DYNLL2, as an MBNL1-interacting protein and demonstrate that their interaction is RNA-independent. Using time-lapse imaging, we show that overexpressed MBNL1 and DYNLL2 move along axonal processes together and that MBNL1-knockdown impairs the motility of mCherry-tagged DYNLL2, resulting in a reduced percentage of retrograde DYNLL2 movement. Examination of the distribution of DYNLL2 and activated phospho-TrkB (pTrkB) receptor in EpA960/CaMKII-Cre brains revealed an increase in the postsynaptic membrane fraction (LP1), indicating impaired retrograde transport. Finally, our neuropathological analysis of postmortem DM1 tissue reveals that reduced cytoplasmic MBNL1 expression is associated with an increase in DYNLL2 and activated pTrkB receptor levels in the synaptosomal fraction. Together, our results support that impaired MBNL1-mediated retrograde BDNF-TrkB signaling may contribute to the histopathological phenotypes of DM1., (© 2023. The Author(s).)

Cytoskeleton organization and dynamics are rapidly regulated by post-translational modifications of key target proteins. Acting downstream of the Cdc42 GTPase, the myotonic dystrophy-related Cdc42-binding kinases MRCKα, MRCKβ, and MRCKγ have recently emerged as important players in cytoskeleton regulation through the phosphorylation of proteins such as the regulatory myosin light chain proteins. Compared with the closely related Rho-associated coiled-coil kinases 1 and 2 (ROCK1 and ROCK2), the contributions of the MRCK kinases are less well characterized, one reason for this being that the discovery of potent and selective MRCK pharmacological inhibitors occurred many years after the discovery of ROCK inhibitors. The disclosure of inhibitors, such as BDP5290 and BDP9066, that have marked selectivity for MRCK over ROCK, as well as the dual ROCK + MRCK inhibitor DJ4, has expanded the repertoire of chemical biology tools to study MRCK function in normal and pathological conditions. Recent research has used these novel inhibitors to establish the role of MRCK signalling in epithelial polarization, phagocytosis, cytoskeleton organization, cell motility, and cancer cell invasiveness. Furthermore, pharmacological MRCK inhibition has been shown to elicit therapeutically beneficial effects in cell-based and in vivo studies of glioma, skin, and ovarian cancers.

Myotonic dystrophy type 1 (DM1) is an autosomal dominant hereditary disease caused by abnormal expansion of unstable CTG repeats in the 3' untranslated region of the myotonic dystrophy protein kinase ( DMPK ) gene. This disease mainly affects skeletal muscle, resulting in myotonia, progressive distal muscle weakness, and atrophy, but also affects other tissues and systems, such as the heart and central nervous system. Despite some studies reporting therapeutic strategies for DM1, many issues remain unsolved, such as the contribution of metabolic and mitochondrial dysfunctions to DM1 pathogenesis. Therefore, it is crucial to identify molecular target candidates associated with metabolic processes for DM1. In this study, resorting to a bibliometric analysis, articles combining DM1, and metabolic/metabolism terms were identified and further analyzed using an unbiased strategy of automatic text mining with VOSviewer software. A list of candidate molecular targets for DM1 associated with metabolic/metabolism was generated and compared with genes previously associated with DM1 in the DisGeNET database. Furthermore, g:Profiler was used to perform a functional enrichment analysis using the Gene Ontology (GO) and REAC databases. Enriched signaling pathways were identified using integrated bioinformatics enrichment analyses. The results revealed that only 15 of the genes identified in the bibliometric analysis were previously associated with DM1 in the DisGeNET database. Of note, we identified 71 genes not previously associated with DM1, which are of particular interest and should be further explored. The functional enrichment analysis of these genes revealed that regulation of cellular metabolic and metabolic processes were the most associated biological processes. Additionally, a number of signaling pathways were found to be enriched, e.g., signaling by receptor tyrosine kinases, signaling by NRTK1 (TRKA), TRKA activation by NGF, PI3K-AKT activation, prolonged ERK activation events, and axon guidance. Overall, several valuable target candidates related to metabolic processes for DM1 were identified, such as NGF, NTRK1, RhoA , ROCK1 , ROCK2, DAG, ACTA, ID1, ID2 MYOD , and MYOG . Therefore, our study strengthens the hypothesis that metabolic dysfunctions contribute to DM1 pathogenesis, and the exploitation of metabolic dysfunction targets is crucial for the development of future therapeutic interventions for DM1.

Myotonic dystrophy, or dystrophia myotonica type 1 (DM1), is a multi-systemic disorder and is the most common adult form of muscular dystrophy. It affects not only muscles but also many organs, including the brain. Cerebral impairments include cognitive deficits, daytime sleepiness, and loss of visuospatial and memory functions. The expression of mutated transcripts with CUG repeats results in a gain of toxic mRNA function. The antisense oligonucleotide (ASO) strategy to treat DM1 brain deficits is limited by the fact that ASOs do not cross the blood-brain barrier after systemic administration, indicating that other methods of delivery should be considered. ASO technology has emerged as a powerful tool for developing potential new therapies for a wide variety of human diseases, and its potential has been proven in a recent clinical trial. Targeting DMPK mRNA in neural cells derived from human induced pluripotent stem cells obtained from a DM1 patient with the IONIS 486178 ASO abolished CUG-expanded foci, enabled nuclear redistribution of MBNL1/2, and corrected aberrant splicing. Intracerebroventricular injection of the IONIS 486178 ASO in DMSXL mice decreased the levels of mutant DMPK mRNAs by up to 70% throughout different brain regions. It also reversed behavioral abnormalities following neonatal administration. The present study indicated that the IONIS 486178 ASO targets mutant DMPK mRNAs in the brain and strongly supports the feasibility of a therapy for DM1 patients based on the intrathecal injection of an ASO., (© 2022. The Author(s).)

Phagocytosis requires actin dynamics, but whether actomyosin contractility plays a role in this morphodynamic process is unclear. Here, we show that in the retinal pigment epithelium (RPE), particle binding to Mer Tyrosine Kinase (MerTK), a widely expressed phagocytic receptor, stimulates phosphorylation of the Cdc42 GEF Dbl3, triggering activation of MRCKβ/myosin-II and its coeffector N-WASP, membrane deformation, and cup formation. Continued MRCKβ/myosin-II activity then drives recruitment of a mechanosensing bridge, enabling cytoskeletal force transmission, cup closure, and particle internalization. In vivo, MRCKβ is essential for RPE phagocytosis and retinal integrity. MerTK-independent activation of MRCKβ signaling by a phosphomimetic Dbl3 mutant rescues phagocytosis in retinitis pigmentosa RPE cells lacking functional MerTK. MRCKβ is also required for efficient particle translocation from the cortex into the cell body in Fc receptor-mediated phagocytosis. Thus, conserved MRCKβ signaling at the cortex controls spatiotemporal regulation of actomyosin contractility to guide distinct phases of phagocytosis in the RPE and represents the principle phagocytic effector pathway downstream of MerTK., (© 2022 Zihni et al.)

RNA regulation in mammalian cells requires complex physical compartmentalisation, using structures thought to be formed by liquid-liquid phase separation. Disruption of these structures is implicated in numerous degenerative diseases. Myotonic dystrophy type 1 (DM1) is a multi-systemic trinucleotide repeat disorder resulting from an expansion of nucleotides CTG (CTGexp) in the DNA encoding DM1 protein kinase (DMPK). The cellular hallmark of DM1 is the formation of nuclear foci that contain expanded DMPK RNA (CUGexp) (with thymine instead of uracil). We report here the deregulation of stress granules (SGs) and processing bodies (P-bodies), two cytoplasmic structures key for mRNA regulation, in cell culture models of DM1. Alterations to the rates of formation and dispersal of SGs suggest an altered ability of cells to respond to stress associated with DM1, while changes to the structure and dynamics of SGs and P-bodies suggest that a widespread alteration to the biophysical properties of cellular structures is a consequence of the presence of CUGexp RNA., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Myotonic dystrophy type 1 (DM1) is the most common muscular dystrophy affecting many different body tissues, predominantly skeletal and cardiac muscles and the central nervous system. The expansion of CTG repeats in the DM1 protein-kinase ( DMPK ) gene is the genetic cause of the disease. The pathogenetic mechanisms are mainly mediated by the production of a toxic expanded CUG transcript from the DMPK gene. With the availability of new knowledge, disease models, and technical tools, much progress has been made in the discovery of altered pathways and in the potential of therapeutic intervention, making the path to the clinic a closer reality. In this review, we describe and discuss the molecular therapeutic strategies for DM1, which are designed to directly target the CTG genomic tract, the expanded CUG transcript or downstream signaling molecules.

ABCB4, is an adenosine triphosphate-binding cassette (ABC) transporter localized at the canalicular membrane of hepatocytes, where it mediates phosphatidylcholine secretion into bile. Gene variations of ABCB4 cause different types of liver diseases, including progressive familial intrahepatic cholestasis type 3 (PFIC3). The molecular mechanisms underlying the trafficking of ABCB4 to and from the canalicular membrane are still unknown. We identified the serine/threonine kinase Myotonic dystrophy kinase-related Cdc42-binding kinase isoform α (MRCKα) as a novel partner of ABCB4. The role of MRCKα was explored, either by expression of dominant negative mutant or by gene silencing using the specific RNAi and CRISPR-cas9 strategy in cell models. The expression of a dominant-negative mutant of MRCKα and MRCKα inhibition by chelerythrine both caused a significant increase in ABCB4 steady-state expression in primary human hepatocytes and HEK-293 cells. RNA interference and CRISPR-Cas9 knockout of MRCKα also caused a significant increase in the amount of ABCB4 protein expression. We demonstrated that the effect of MRCKα was mediated by its downstream effector, the myosin II regulatory light chain (MRLC), which was shown to also bind ABCB4. Our findings provide evidence that MRCKα and MRLC bind to ABCB4 and regulate its cell surface expression.

Skeletal muscle tissue is severely affected in myotonic dystrophy type 1 (DM1) patients, characterised by muscle weakness, myotonia and muscle immaturity in the most severe congenital form of the disease. Previously, it was not known at what stage during myogenesis the DM1 phenotype appears. In this study we differentiated healthy and DM1 human embryonic stem cells to myoblasts and myotubes and compared their differentiation potential using a comprehensive multi-omics approach. We found myogenesis in DM1 cells to be abnormal with altered myotube generation compared to healthy cells. We did not find differentially expressed genes between DM1 and non-DM1 cell lines within the same developmental stage. However, during differentiation we observed an aberrant inflammatory response and increased CpG methylation upstream of the CTG repeat at the myoblast level and RNA mis-splicing at the myotube stage. We show that early myogenesis modelled in hESC reiterates the early developmental manifestation of DM1., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Myotonic dystrophy type 1 (DM1) is a hereditary and multisystemic disease characterized by myotonia, progressive distal muscle weakness and atrophy. The molecular mechanisms underlying this disease are still poorly characterized, although there are some hypotheses that envisage to explain the multisystemic features observed in DM1. An emergent hypothesis is that nuclear envelope (NE) dysfunction may contribute to muscular dystrophies, particularly to DM1. Therefore, the main objective of the present study was to evaluate the nuclear profile of DM1 patient-derived and control fibroblasts and to determine the protein levels and subcellular distribution of relevant NE proteins in these cell lines. Our results demonstrated that DM1 patient-derived fibroblasts exhibited altered intracellular protein levels of lamin A/C, LAP1, SUN1, nesprin-1 and nesprin-2 when compared with the control fibroblasts. In addition, the results showed an altered location of these NE proteins accompanied by the presence of nuclear deformations (blebs, lobes and/or invaginations) and an increased number of nuclear inclusions. Regarding the nuclear profile, DM1 patient-derived fibroblasts had a larger nuclear area and a higher number of deformed nuclei and micronuclei than control-derived fibroblasts. These results reinforce the evidence that NE dysfunction is a highly relevant pathological characteristic observed in DM1.

Myotonic dystrophy is a dominantly inherited multisystem disorder that results from increased CTG repeats in the 3' region of the myotonic dystrophy protein kinase gene (DMPK). The mutant DMPK mRNA remains in the nucleus and sequesters RNA-binding proteins, including regulators of mRNA splicing. Myotonic dystrophy is characterized by a highly variable phenotype that includes muscle weakness and myotonia, and the disorder may affect the function of many endocrine glands. DMPK mRNA is expressed in muscle, testis, liver, pituitary, thyroid, and bone; the mutated form leads to disruption of meiosis and an increase in fetal insulin receptor-A relative to adult insulin receptor-B, resulting in adult primary testicular failure and insulin resistance predisposing to diabetes, respectively. Patients with myotonic dystrophy are also at increased risk for hyperlipidemia, nonalcoholic fatty liver disease, erectile dysfunction, benign and malignant thyroid nodules, bone fractures, miscarriage, preterm delivery, and failed labor during delivery. Circulating parathyroid hormone and adrenocorticotropic hormone levels may be elevated, but the mechanisms for these associations are unclear. This review summarizes what is known about endocrine dysfunction in individuals with myotonic dystrophy., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

MRCKα is a ubiquitously expressed serine/threonine kinase involved in cell contraction and F-actin turnover, which is highly amplified in human breast cancer and part of a gene expression signature for bad prognosis. Nothing is known about the in vivo function of MRCKα. To explore MRCKα function in development and in breast cancer, we generated mice lacking a functional MRCKα gene. Mice were born close to the Mendelian ratio and showed no obvious phenotype including a normal mammary gland formation. Assessing breast cancer development using the transgenic MMTV-PyMT mouse model, loss of MRCKα did not affect tumor onset, tumor growth and metastasis formation. Deleting MRCKα and its related family member MRCKβ in two triple-negative breast cancer cell lines resulted in reduced invasion of MDA-MB-231 cells, but did not affect migration of 4T1 cells. Further genomic analysis of human breast cancers revealed that MRCKα is frequently co-amplified with the oncogenes ARID4B and AKT3 which might contribute to the prognostic value of MRCKα expression. Collectively, these data suggest that MRCKα might be a prognostic marker for breast cancer, but probably of limited functional importance.

An intact lung epithelial barrier is essential for lung homeostasis. The Na+, K+-ATPase (NKA), primarily serving as an ion transporter, also regulates epithelial barrier function via modulation of tight junctions. However, the underlying mechanism is not well understood. Here, we show that overexpression of the NKA β1 subunit upregulates the expression of tight junction proteins, leading to increased alveolar epithelial barrier function by an ion transport-independent mechanism. Using IP and mass spectrometry, we identified a number of unknown protein interactions of the β1 subunit, including a top candidate, myotonic dystrophy kinase-related cdc42-binding kinase α (MRCKα), which is a protein kinase known to regulate peripheral actin formation. Using a doxycycline-inducible gene expression system, we demonstrated that MRCKα and its downstream activation of myosin light chain is required for the regulation of alveolar barrier function by the NKA β1 subunit. Importantly, MRCKα is expressed in both human airways and alveoli and has reduced expression in patients with acute respiratory distress syndrome (ARDS), a lung illness that can be caused by multiple direct and indirect insults, including the infection of influenza virus and SARS-CoV-2. Our results have elucidated a potentially novel mechanism by which NKA regulates epithelial tight junctions and have identified potential drug targets for treating ARDS and other pulmonary diseases that are caused by barrier dysfunction.

Background: Helicobacter pylori-mediated gastric carcinogenesis is initiated by a plethora of signaling events in the infected gastric epithelial cells (GECs). The E3 ubiquitin ligase seven in absentia homolog 2 (Siah2) is induced in GECs in response to H. pylori infection. Posttranslational modifications of Siah2 orchestrate its function as well as stability. The aim of this study was to evaluate Siah2 phosphorylation status under the influence of H. pylori infection and its impact in gastric cancer progression., Methods: H. pylori-infected various GECs, gastric tissues from H. pylori-infected GC patients and H. felis-infected C57BL/6 mice were evaluated for Siah2 phosphorylation by western blotting or immunofluorescence microscopy. Coimmunoprecipitation assay followed by mass spectrometry were performed to identify the kinases interacting with Siah2. Phosphorylation sites of Siah2 were identified by using various plasmid constructs generated by site-directed mutagenesis. Proteasome inhibitor MG132 was used to investigate proteasome degradation events. The importance of Siah2 phosphorylation on tumorigenicity of infected cells were detected by using phosphorylation-null mutant and wild type Siah2 stably-transfected cells followed by clonogenicity assay, cell proliferation assay, anchorage-independent growth and transwell invasion assay., Results: Siah2 was phosphorylated in H. pylori-infected GECs as well as in metastatic GC tissues at residues serine 6  (Ser 6 ) and threonine 279  (Thr 279 ). Phosphorylation of Siah2 was mediated by MRCKβ, a Ser/Thr protein kinase. MRCKβ was consistently expressed in uninfected GECs and noncancer gastric tissues but its level decreased in infected GECs as well as in metastatic tissues which had enhanced Siah2 expression. Infected murine gastric tissues showed similar results. MRCKβ could phosphorylate Siah2 but itself got ubiquitinated from this interaction leading to the proteasomal degradation of MRCKβ and use of proteasomal inhibitor MG132 could rescue MRCKβ from Siah2-mediated degradation. Ser 6 and Thr 279 phosphorylated-Siah2 was more stable and tumorigenic than its non-phosphorylated counterpart as revealed by the proliferation, invasion, migration abilities and anchorage-independent growth of stable-transfected cells., Conclusions: Increased level of Ser 6 and Thr 279 -phosphorylated-Siah2 and downregulated MRCKβ were prominent histological characteristics of Helicobacter-infected gastric epithelium and metastatic human GC. MRCKβ-dependent Siah2 phosphorylation stabilized Siah2 which promoted anchorage-independent survival and proliferative potential of GECs. Phospho-null mutants of Siah2 (S6A and T279A) showed abated tumorigenicity.

Myotonic dystrophy type 1 (DM1) is the most common monogenetic muscular disorder of adulthood. This multisystemic disease is caused by CTG repeat expansion in the 3'-untranslated region of the DM1 protein kinase gene called DMPK. DMPK encodes a myosin kinase expressed in skeletal muscle cells and other cellular populations such as smooth muscle cells, neurons and fibroblasts. The resultant expanded (CUG)n RNA transcripts sequester RNA binding factors leading to ubiquitous and persistent splicing deregulation. The accumulation of mutant CUG repeats is linked to increased activity of glycogen synthase kinase 3 beta (GSK3β), a highly conserved and ubiquitous serine/threonine kinase with functions in pathways regulating inflammation, metabolism, oncogenesis, neurogenesis and myogenesis. As GSK3β-inhibition ameliorates defects in myogenesis, muscle strength and myotonia in a DM1 mouse model, this kinase represents a key player of DM1 pathobiochemistry and constitutes a promising therapeutic target. To better characterise DM1 patients, and monitor treatment responses, we aimed to define a set of robust disease and severity markers linked to GSK3βby unbiased proteomic profiling utilizing fibroblasts derived from DM1 patients with low (80- 150) and high (2600- 3600) CTG-repeats. Apart from GSK3β increase, we identified dysregulation of nine proteins (CAPN1, CTNNB1, CTPS1, DNMT1, HDAC2, HNRNPH3, MAP2K2, NR3C1, VDAC2) modulated by GSK3β. In silico-based expression studies confirmed expression in neuronal and skeletal muscle cells and revealed a relatively elevated abundance in fibroblasts. The potential impact of each marker in the myopathology of DM1 is discussed based on respective function to inform potential uses as severity markers or for monitoring GSK3β inhibitor treatment responses.

The CDC42 small GTPase is a major influence on actin-myosin cytoskeleton organization and dynamics, signalling via effector proteins including the M yotonic dystrophy r elated C DC42-binding protein k inases (MRCK) α and β. We previously identified Serine 1003 of MRCKα as a site of autophosphorylation, and showed that a phosphorylation-sensitive antibody raised against this site could be used as a surrogate indicator of kinase activity. In this study, a kinase-dead version of MRCKβ was established by mutation of the conserved Lysine 105 to Methionine (K105M), which was then used for mass spectrometry analysis to identify phosphorylation events that occurred in catalytically-competent MRCKβ but not in the kinase-dead form. A total of ten phosphorylations were identified on wild-type MRCKβ, of which the previously undescribed Threonine 1108 (Thr1108) was not found on kinase-dead MRCKβ K105M, consistent with this being due to autophosphorylation. Mutation of Thr1108 to non-phosphorylatable Alanine (T1108A) or phosphomimetic Glutamate (T1108E) did not affect the ability of MRCKβ to phosphorylate recombinant myosin light chain in vitro , or observably alter the subcellular localization of green fluorescent protein (GFP)-tagged MRCKβ expressed in MDA MB 231 human breast cancer cells. Although phosphorylation of Thr1108 did not appear to contribute to MRCKβ function or regulation, the identification of this phosphorylation does make it possible to characterize whether this site could be used as a surrogate biomarker of kinase activity and inhibitor efficacy as we previously demonstrated for Ser 1003 in MRCKα.

Targeted protein degraders are an emerging modality. Their properties fall outside the traditional small-molecule property space and are in the 'beyond rule of 5' space. Consequently, drug discovery programs focused on developing orally bioavailable degraders are expected to face complex drug metabolism and pharmacokinetics (DMPK) challenges compared with traditional small molecules. Nevertheless, little information is available on the DMPK optimization of oral degraders. Therefore, in this review, we discuss our experience of these DMPK optimization challenges and present methodologies and strategies to overcome the hurdles dealing with this new small-molecule modality, specifically in developing oral degraders to treat cancer., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Expansion of an unstable CTG repeat in the 3'UTR of the DMPK gene causes Myotonic Dystrophy type 1 (DM1). CUG-expanded DMPK transcripts (CUGexp) sequester Muscleblind-like (MBNL) alternative splicing regulators in ribonuclear inclusions (foci), leading to abnormalities in RNA processing and splicing. To alleviate the burden of CUGexp, we tested therapeutic approach utilizing antisense oligonucleotides (AONs)-mediated DMPK splice-switching and degradation of mutated pre-mRNA. Experimental design involved: (i) skipping of selected constitutive exons to induce frameshifting and decay of toxic mRNAs by an RNA surveillance mechanism, and (ii) exclusion of the alternative exon 15 (e15) carrying CUGexp from DMPK mRNA. While first strategy failed to stimulate DMPK mRNA decay, exclusion of e15 enhanced DMPK nuclear export but triggered accumulation of potentially harmful spliced out pre-mRNA fragment containing CUGexp. Neutralization of this fragment with antisense gapmers complementary to intronic sequences preceding e15 failed to diminish DM1-specific spliceopathy due to AONs' chemistry-related toxicity. However, intronic gapmers alone reduced the level of DMPK mRNA and mitigated DM1-related cellular phenotypes including spliceopathy and nuclear foci. Thus, a combination of the correct chemistry and experimental approach should be carefully considered to design a safe AON-based therapeutic strategy for DM1., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

High-grade serous ovarian carcinoma (HGSOC) is the most lethal gynecological cancer with few effective, targeted therapies. HGSOC tumors exhibit genomic instability with frequent alterations in the protein kinome; however, only a small fraction of the kinome has been therapeutically targeted in HGSOC. Using multiplexed inhibitor beads and mass spectrometry, we mapped the kinome landscape of HGSOC tumors from patients and patient-derived xenograft models. The data revealed a prevalent signature consisting of established HGSOC driver kinases, as well as several kinases previously unexplored in HGSOC. Loss-of-function analysis of these kinases in HGSOC cells indicated MRCKA (also known as CDC42BPA) as a putative therapeutic target. Characterization of the effects of MRCKA knockdown in established HGSOC cell lines demonstrated that MRCKA was integral to signaling that regulated the cell cycle checkpoint, focal adhesion, and actin remodeling, as well as cell migration, proliferation, and survival. Moreover, inhibition of MRCKA using the small-molecule BDP9066 decreased cell proliferation and spheroid formation and induced apoptosis in HGSOC cells, suggesting that MRCKA may be a promising therapeutic target for the treatment of HGSOC., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Myotonic dystrophy type 1 (DM1) is a complex neuromuscular disease caused by an unstable cardiotocography (CTG) repeat expansion in the DMPK gene. This disease is characterized by high clinical and genetic variability, leading to some difficulties in the diagnosis and prognosis of DM1. Better understanding the origin of this variability is important for developing new challenging therapies and, in particular, for progressing on the path of personalized treatments. Here, we reviewed CTG triplet repeat instability and its modifiers as an important source of phenotypic variability in patients with DM1., Competing Interests: The authors no conflict of interest.

Myotonic dystrophy type 1 (DM1) is a complex genetic disease affecting many tissues. DM1 is caused by an expansion of CTG repeats in the 3'-UTR of the DMPK gene. The mechanistic studies of DM1 suggested that DMPK mRNA, containing expanded CUG repeats, is a major therapeutic target in DM1. Therefore, the removal of the toxic RNA became a primary focus of the therapeutic development in DM1 during the last decade. However, a cure for this devastating disease has not been found. Whereas the degradation of toxic RNA remains a preferential approach for the reduction of DM1 pathology, other approaches targeting early toxic events downstream of the mutant RNA could be also considered. In this review, we discuss the beneficial role of the restoring of the RNA-binding protein, CUGBP1/CELF1, in the correction of DM1 pathology. It has been recently found that the normalization of CUGBP1 activity with the inhibitors of GSK3 has a positive effect on the reduction of skeletal muscle and CNS pathologies in DM1 mouse models. Surprisingly, the inhibitor of GSK3, tideglusib also reduced the toxic CUG-containing RNA. Thus, the development of the therapeutics, based on the correction of the GSK3β-CUGBP1 pathway, is a promising option for this complex disease.

Short tandem repeats (STRs) are prone to expansion mutations that cause multiple hereditary neurological and neuromuscular diseases. To study pathomechanisms using mouse models that recapitulate the tissue specificity and developmental timing of an STR expansion gene, we used rolling circle amplification and CRISPR/Cas9-mediated genome editing to generate Dmpk CTG expansion (CTG exp ) knockin models of myotonic dystrophy type 1 (DM1). We demonstrate that skeletal muscle myoblasts and brain choroid plexus epithelial cells are particularly susceptible to Dmpk CTG exp mutations and RNA missplicing. Our results implicate dysregulation of muscle regeneration and cerebrospinal fluid homeostasis as early pathogenic events in DM1., (© 2019 Nutter et al.; Published by Cold Spring Harbor Laboratory Press.)

Antisense oligonucleotides (ASOs) targeting pathologic RNAs have shown promising therapeutic corrections for many genetic diseases including myotonic dystrophy (DM1). Thus, ASO strategies for DM1 can abolish the toxic RNA gain-of-function mechanism caused by nucleus-retained mutant DMPK (DM1 protein kinase) transcripts containing CUG expansions (CUGexps). However, systemic use of ASOs for this muscular disease remains challenging due to poor drug distribution to skeletal muscle. To overcome this limitation, we test an arginine-rich Pip6a cell-penetrating peptide and show that Pip6a-conjugated morpholino phosphorodiamidate oligomer (PMO) dramatically enhanced ASO delivery into striated muscles of DM1 mice following systemic administration in comparison with unconjugated PMO and other ASO strategies. Thus, low-dose treatment with Pip6a-PMO-CAG targeting pathologic expansions is sufficient to reverse both splicing defects and myotonia in DM1 mice and normalizes the overall disease transcriptome. Moreover, treated DM1 patient-derived muscle cells showed that Pip6a-PMO-CAG specifically targets mutant CUGexp-DMPK transcripts to abrogate the detrimental sequestration of MBNL1 splicing factor by nuclear RNA foci and consequently MBNL1 functional loss, responsible for splicing defects and muscle dysfunction. Our results demonstrate that Pip6a-PMO-CAG induces long-lasting correction with high efficacy of DM1-associated phenotypes at both molecular and functional levels, and strongly support the use of advanced peptide conjugates for systemic corrective therapy in DM1.

A CTG repeat expansion in the DMPK gene is the causative mutation of myotonic dystrophy type 1 (DM1). Transcription of the expanded CTG repeat produces toxic gain-of-function CUG RNA, leading to disease symptoms. A screening platform that targets production or stability of the toxic CUG RNA in a selective manner has the potential to provide new biological and therapeutic insights. A DM1 HeLa cell model was generated that stably expresses a toxic r(CUG)480 and an analogous r(CUG)0 control from DMPK and was used to measure the ratio-metric level of r(CUG)480 versus r(CUG)0. This DM1 HeLa model recapitulates pathogenic hallmarks of DM1, including CUG ribonuclear foci and missplicing of pre-mRNA targets of the muscleblind (MBNL) alternative splicing factors. Repeat-selective screening using this cell line led to the unexpected identification of multiple microtubule inhibitors as hits that selectively reduce r(CUG)480 levels and partially rescue MBNL-dependent missplicing. These results were validated by using the Food and Drug Administration-approved clinical microtubule inhibitor colchicine in DM1 mouse and primary patient cell models. The mechanism of action was found to involve selective reduced transcription of the CTG expansion that we hypothesize to involve the LINC (linker of nucleoskeleton and cytoskeleton) complex. The unanticipated identification of microtubule inhibitors as selective modulators of toxic CUG RNA opens research directions for this form of muscular dystrophy and may shed light on the biology of CTG repeat expansion and inform therapeutic avenues. This approach has the potential to identify modulators of expanded repeat-containing gene expression for over 30 microsatellite expansion disorders., Competing Interests: The authors declare no competing interest.

Tumor suppressor p53 plays an integral role in DNA-damage induced apoptosis, a biological process that protects against tumor progression. Cell shape dramatically changes when cells undergo apoptosis, which is associated with actomyosin contraction; however, it remains entirely elusive how p53 regulates actomyosin contraction in response to DNA-damaging agents. To identify a novel p53 regulating gene encoding the modulator of myosin, we conducted DNA microarray analysis. We found that, in response to DNA-damaging agent doxorubicin, expression of myotonic dystrophy protein kinase (DMPK), which is known to upregulate actomyosin contraction, was increased in a p53-dependent manner. The promoter region of DMPK gene contained potential p53-binding sequences and its promoter activity was increased by overexpression of the p53 family protein p73, but, unexpectedly, not of p53. Furthermore, we found that doxorubicin treatment induced p73 expression, which was significantly attenuated by downregulation of p53. These data suggest that p53 induces expression of DMPK through upregulating p73 expression. Overexpression of DMPK promotes contraction of the actomyosin cortex, which leads to formation of membrane blebs, loss of cell adhesion, and concomitant caspase activation. Taken together, our results suggest the existence of p53-p73-DMPK axis which mediates DNA-damage induced actomyosin contraction at the cortex and concomitant cell death.

Apoptosis plays a crucial role in clearing old or critically compromised cells, and actively maintains epithelial homeostasis and epithelial morphogenesis during embryo development. But how is the apoptotic signaling pathway able to orchestrate such complex and dynamic multi-cellular morphological events at the tissue scale? In this review we collected the most updated knowledge regarding how apoptosis controls different cytoskeletal components. We describe how apoptosis can control epithelial homeostasis though epithelial extrusion, a highly orchestrated process based on high- order actomyosin structures and on the coordination between the apoptotic and the neighboring cells. Finally, we describe how the synergy among forces generated by multiple apoptotic cells can shape epithelia in embryo development.

Myotonic dystrophy type 1 (DM1) is a complex neuromuscular disorder caused by expansion of a CTG repeat in the 3'-untranslated region (UTR) of the DMPK gene. Mutant DMPK transcripts form aberrant structures and anomalously associate with RNA-binding proteins (RBPs). As a first step toward better understanding of the involvement of abnormal DMPK mRNA folding in DM1 manifestation, we used SHAPE, DMS, CMCT, and RNase T1 structure probing in vitro for modeling of the topology of the DMPK 3'-UTR with normal and pathogenic repeat lengths of up to 197 CUG triplets. The resulting structural information was validated by disruption of base-pairing with LNA antisense oligonucleotides (AONs) and used for prediction of therapeutic AON accessibility and verification of DMPK knockdown efficacy in cells. Our model for DMPK RNA structure demonstrates that the hairpin formed by the CUG repeat has length-dependent conformational plasticity, with a structure that is guided by and embedded in an otherwise rigid architecture of flanking regions in the DMPK 3'-UTR. Evidence is provided that long CUG repeats may form not only single asymmetrical hairpins but also exist as branched structures. These newly identified structures have implications for DM1 pathogenic mechanisms, like sequestration of RBPs and repeat-associated non-AUG (RAN) translation., (© 2019 van Cruchten et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

This letter describes the first account of the chemical optimization (SAR and DMPK profiling) of a new series of mGlu 4 positive allosteric modulators (PAMs), leading to the identification of VU0652957 (VU2957, Valiglurax), a compound profiled as a preclinical development candidate. Here, we detail the challenges faced in allosteric modulator programs (e.g., steep SAR, as well as subtle structural changes affecting overall physiochemical/DMPK properties and CNS penetration)., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objectives: In this study, we aim to determine how CUG-expansion and the abundance of Celf1 regulates normal myocyte differentiation and reveal the role ofmiR-206 in myotonic dystrohy and explore a possible gene therapy vector. Methods:  we set up CUG-expansion and Celf1 overexpression C2C12 cell models to imitate the myocyte differentiation defects of DM1, then transfected AdvmiR-206 into cell models, tested the level of myogenic markers MyoD, MyoG, Mef2c, Celf1 by RT-PCRand Western Blotting, detected myotube formation by myosin heavy chain immunostaining.  Result:  3'-UTR CUG-expansion leads to myotube defects and impaired myoblasts differentiation. Overexpression of Celf1 inhibits myoblast differentiation and impairs differentiation. Knockdown of Celf1 partially rescues differentiation defects of myoblasts harboring CUG-expansion. miR-206 incompletely reverses myoblast differentiation inhibition induced by CUG-expansion and partially recuses myoblast differentiation defects induced by Celf1 overexpression. Conclusions: Ectopic miR-206 mimicking the endogenous temporal patterns specifically drives a myocyte program that boosts myoblast lineages, likely by promoting the expression of MyoD to rectify the myogenic deficiency by stimulating the accumulation of Celf1. Abbreviations : DMPK: (dystrophia myotonica protein kinase); 3'-UTR: (3'-untranslated region); MBNL1: (muscleblind-like [Drosophila]); DM1: (myotonic dystrophy type 1); GFP: (green fluorescent protein); RT-PCR: (quantitative reverse transcriptase-polymerase chain reaction); shRNA: (short hairpin RNA).

Kinase enzymes phosphorylate protein substrates in a highly ordered manner to control cell signaling. Unregulated kinase activity is associated with a variety of disease states, most notably cancer, making the characterization of kinase activity in cells critical to understand disease formation. However, the paucity of available tools has prevented a full mapping of the substrates and interacting proteins of kinases involved in cellular function. Recently we developed kinase-catalyzed cross-linking to covalently connect substrate and kinase in a phosphorylation-dependent manner. Here, we report a new method combining kinase-catalyzed cross-linking and immunoprecipitation (K-CLIP) to identify kinase-substrate pairs and kinase-associated proteins. K-CLIP was applied to the substrate p53, which is robustly phosphorylated. Both known and unknown kinases of p53 were isolated from cell lysates using K-CLIP. In follow-up validation studies, MRCKbeta was identified as a new p53 kinase. Beyond kinases, a variety of p53 and kinase-associated proteins were also identified using K-CLIP, which provided a snapshot of cellular interactions. The K-CLIP method represents an immediately useful chemical tool to identify kinase-substrate pairs and multiprotein complexes in cells, which will embolden cell signaling research and enhance our understanding of kinase activity in normal and disease states.

Deep layer III pyramidal cells in the dorsolateral prefrontal cortex (DLPFC) from subjects with schizophrenia and bipolar disorder previously were shown to exhibit dendritic arbor pathology. This study sought to determine whether MARCKS, its regulatory protein dysbindin-1, and two proteins, identified using microarray data, CDC42BPA and ARHGEF6, were associated with dendritic arbor pathology in the DLPFC from schizophrenia and bipolar disorder subjects. Using western blotting, relative protein expression was assessed in the DLPFC (BA 46) grey matter from subjects with schizophrenia (n = 19), bipolar disorder (n = 17) and unaffected control subjects (n = 19). Protein expression data were then correlated with dendritic parameter data obtained previously. MARCKS and dysbindin-1a expression levels did not differ among the three groups. Dysbindin-1b expression was 26% higher in schizophrenia subjects (p = 0.01) and correlated inversely with basilar dendrite length (r = -0.31, p = 0.048) and the number of spines per basilar dendrite (r = -0.31, p = 0.048), but not with dendritic spine density (r = -0.16, p = 0.32). The protein expression of CDC42BPA was 33% higher in schizophrenia subjects (p = 0.03) but, did not correlate with any dendritic parameter (p > 0.05). ARHGEF6 87 kDa isoform expression did not differ among the groups. CDC42BPA expression was not altered in frontal cortex from rats chronically administered haloperidol or clozapine. Dysbindin-1b appears to play a role in dendritic arbor pathology observed previously in the DLPFC in schizophrenia., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Aims: The pathology of heart failure is characterized by poorly contracting and dilated ventricles. At the cellular level, this is associated with lengthening of individual cardiomyocytes and loss of sarcomeres. While it is known that the transcription factor myocyte enhancer factor-2 (MEF2) is involved in this cardiomyocyte remodelling, the underlying mechanism remains to be elucidated. Here, we aim to mechanistically link MEF2 target genes with loss of sarcomeres during cardiomyocyte remodelling., Methods and Results: Neonatal rat cardiomyocytes overexpressing MEF2 elongated and lost their sarcomeric structure. We identified myotonic dystrophy protein kinase (DMPK) as direct MEF2 target gene involved in this process. Adenoviral overexpression of DMPK E, the isoform upregulated in heart failure, resulted in severe loss of sarcomeres in vitro, and transgenic mice overexpressing DMPK E displayed disruption of sarcomere structure and cardiomyopathy in vivo. Moreover, we found a decreased expression of sarcomeric genes following DMPK E gain-of-function. These genes are targets of the transcription factor serum response factor (SRF) and we found that DMPK E acts as inhibitor of SRF transcriptional activity., Conclusion: Our data indicate that MEF2-induced loss of sarcomeres is mediated by DMPK via a decrease in sarcomeric gene expression by interfering with SRF transcriptional activity. Together, these results demonstrate an unexpected role for DMPK as a direct mediator of adverse cardiomyocyte remodelling and heart failure.

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are human neuromuscular disorders associated with mutations of simple repetitive sequences in affected genes. The abnormal expansion of CTG repeats in the 3'-UTR of the DMPK gene elicits DM1, whereas elongated CCTG repeats in intron 1 of ZNF9/CNBP triggers DM2. Pathogenesis of both disorders is manifested by nuclear retention of expanded repeat-containing RNAs and aberrant alternative splicing. The precise determination of absolute numbers of mutant RNA molecules is important for a better understanding of disease complexity and for accurate evaluation of the efficacy of therapeutic drugs. We present two quantitative methods, Multiplex Ligation-Dependent Probe Amplification and droplet digital PCR, for studying the mutant DMPK transcript (DMPK exp RNA) and the aberrant alternative splicing in DM1 and DM2 human tissues and cells. We demonstrate that in DM1, the DMPK exp RNA is detected in higher copy number than its normal counterpart. Moreover, the absolute number of the mutant transcript indicates its low abundance with only a few copies per cell in DM1 fibroblasts. Most importantly, in conjunction with fluorescence in-situ hybridization experiments, our results suggest that in DM1 fibroblasts, the vast majority of nuclear RNA foci consist of a few molecules of DMPK exp RNA.

Toxic RNAs containing expanded trinucleotide repeats are the cause of many neuromuscular disorders, one being myotonic dystrophy type 1 (DM1). DM1 is triggered by CTG-repeat expansion in the 3'-untranslated region of the DMPK gene, resulting in a toxic gain of RNA function through sequestration of MBNL1 protein, among others. Herein, we report the development of a relatively short miniPEG-γ peptide nucleic acid probe, two triplet repeats in length, containing terminal pyrene moieties, that is capable of binding rCUG repeats in a sequence-specific and selective manner. The newly designed probe can discriminate the pathogenic rCUG exp from the wild-type transcript and disrupt the rCUG exp -MBNL1 complex. The work provides a proof of concept for the development of relatively short nucleic acid probes for targeting RNA-repeat expansions associated with DM1 and other related neuromuscular disorders.

Metastasis is one of the major causes of treatment failure in the patients with colon cancer. The aim of our study is to find key proteins and pathways that drive invasion and metastasis in colon cancer. Eight rounds of selection of cancer cells invading through matrigel-coated chamber were performed to obtain highly invasive colon cancer sublines HCT116-I8 and RKO-I8. Stable Isotope Labeling by Amino Acids in Cell Culture technology was used to identify the differently expressed proteins, and the proteomics data were analyzed by ingenuity pathway analysis. PAK1-PBD immunoprecipitation combined with Western blot were carried out to determine Cdc42 activity, and qRT-PCR and Western blot were used to determine gene expression. The functional role of Cdc42BPA and Cdc42 pathway in colon cancer invasion was studied by loss-of-function experiments including pharmacological blockade, siRNA knockdown, chamber invasion, and WST-1 assays. Human colon cancer tissue microarray was analyzed by immunohistochemistry for overexpression of Cdc42BPA and its correlation with clinicopathological parameters and patient survival outcomes. HCT116-I8 and RKO-I8 cells showed significantly stronger invasive potential as well as decreased E-cadherin and increased vimentin expressions compared with parental cells. The differently expressed proteins in I8 cells compared with parental cells were identified. Bioinformatics analysis of proteomics data suggested that Cdc42BPA protein and Cdc42 signaling pathway are important for colon cancer invasion, which was confirmed by experimental data showing upregulation of Cdc42BPA and higher expression of active GTP-bound form of Cdc42 in HCT116-I8 and RKO-I8 cells. Functionally, pharmacological and genetic blockade of Cdc42BPA and Cdc42 signaling markedly suppressed colon cancer cell invasion and reversed epithelial mesenchymal transition process. Furthermore, compared with adjacent normal tissues, Cdc42BPA expression was significantly higher in colon cancer tissues and further upregulated in metastatic tumors in lymph nodes. More importantly, Cdc42BPA expression was correlated with metastasis and poor survival of the patients with colon cancer. This study provides the first evidence that Cdc42BPA and Cdc42 signaling are important for colon cancer invasion, and Cdc42BPA has potential implications for colon cancer prognosis and treatment.

Extrusion of apoptotic cells from epithelial tissues requires orchestrated morphological rearrangements of the apoptotic cell and its neighbors. However, the connections between the apoptotic cascade and events leading to extrusion are not fully understood. Here, we characterize an apoptotic extrusion apical actin ring (EAAR) that is assembled within the apoptotic cell and drives epithelial extrusion. Caspase-mediated cleavage of myotonic dystrophy kinase-related CDC42-binding kinase-α (MRCKα) triggers a signaling pathway that leads to the assembly of EAAR that pulls actin bundles, resulting in the compaction and removal of the cell body. We provide a detailed portrait of the EAAR including F-actin flow, the contribution of myosin contraction, and actin polymerization at bundles' terminals when the product of MRCKα cleavage is expressed. These results add to our understanding of the mechanisms controlling the process of epithelial extrusion by establishing a causal relationship between the triggering events of apoptosis, the activation of MRCKα, and its subsequent effects on the dynamics of actomyosin cytoskeleton rearrangement., (© 2018 Gagliardi et al.)

Myotonic dystrophy type 1 (DM1) is a multisystemic disease caused by an expanded CTG repeat in the 3' UTR of the dystrophia myotonica protein kinase (DMPK) gene. Short, DNA-based antisense oligonucleotides termed gapmers are a promising strategy to degrade toxic CUG expanded repeat (CUG exp ) RNA. Nucleoside analogs are incorporated to increase gapmer affinity and stability; however, some analogs also exhibit toxicity. In this study, we demonstrate that the 2',4'-BNA NC [NMe] (BNA NC ) modification is a promising nucleoside analog with high potency similar to 2',4'-LNA (LNA). BNA NC gapmers targeting a nonrepetitive region of the DMPK 3' UTR show allele-specific knockdown of CUG exp RNA and revert characteristic DM1 molecular defects including mis-splicing and accumulation of RNA foci. Notably, the BNA NC gapmers tested in this study did not induce caspase activation, in contrast to a sequence matched LNA gapmer. This study indicates that BNA NC gapmers warrant further study as a promising RNA targeting therapeutic.

Polarized epithelia develop distinct cell surface domains, with the apical membrane acquiring characteristic morphological features such as microvilli. Cell polarization is driven by polarity determinants including the evolutionarily conserved partitioning-defective (PAR) proteins that are separated into distinct cortical domains. PAR protein segregation is thought to be a consequence of asymmetric actomyosin contractions. The mechanism of activation of apically polarized actomyosin contractility is unknown. Here we show that the Cdc42 effector MRCK activates myosin-II at the apical pole to segregate aPKC-Par6 from junctional Par3, defining the apical domain. Apically polarized MRCK-activated actomyosin contractility is reinforced by cooperation with aPKC-Par6 downregulating antagonistic RhoA-driven junctional actomyosin contractility, and drives polarization of cytosolic brush border determinants and apical morphogenesis. MRCK-activated polarized actomyosin contractility is required for apical differentiation and morphogenesis in vertebrate epithelia and Drosophila photoreceptors. Our results identify an apical origin of actomyosin-driven morphogenesis that couples cytoskeletal reorganization to PAR polarity signalling.

Myotonic dystrophy Type 1 (DM1) is a rare genetic disease caused by the expansion of CTG trinucleotide repeats ((CTG)exp) in the 3' untranslated region of the DMPK gene. The repeat transcripts sequester the RNA binding protein Muscleblind-like protein 1 (MBNL1) and hamper its normal function in pre-mRNA splicing. Overexpressing exogenous MBNL1 in the DM1 mouse model has been shown to rescue the splicing defects and reverse myotonia. Although a viable therapeutic strategy, pharmacological modulators of MBNL1 expression have not been identified. Here, we engineered a ZsGreen tag into the endogenous MBNL1 locus in HeLa cells and established a flow cytometry-based screening system to identify compounds that increase MBNL1 level. The initial screen of small molecule compound libraries identified more than thirty hits that increased MBNL1 expression greater than double the baseline levels. Further characterization of two hits revealed that the small molecule HDAC inhibitors, ISOX and vorinostat, increased MBNL1 expression in DM1 patient-derived fibroblasts and partially rescued the splicing defect caused by (CUG)exp repeats in these cells. These findings demonstrate the feasibility of this flow-based cytometry screen to identify both small molecule compounds and druggable targets for MBNL1 upregulation., (© The Author 2017. Published by Oxford University Press.)

RNA repeat expansions cause a host of incurable, genetically defined diseases. The most common class of RNA repeats consists of trinucleotide repeats. These long, repeating transcripts fold into hairpins containing 1 × 1 internal loops that can mediate disease via a variety of mechanism(s) in which RNA is the central player. Two of these disorders are Huntington's disease and myotonic dystrophy type 1, which are caused by r(CAG) and r(CUG) repeats, respectively. We report the structures of two RNA constructs containing three copies of a r(CAG) [r(3×CAG)] or r(CUG) [r(3×CUG)] motif that were modeled with nuclear magnetic resonance spectroscopy and simulated annealing with restrained molecular dynamics. The 1 × 1 internal loops of r(3×CAG) are stabilized by one-hydrogen bond (cis Watson-Crick/Watson-Crick) AA pairs, while those of r(3×CUG) prefer one- or two-hydrogen bond (cis Watson-Crick/Watson-Crick) UU pairs. Assigned chemical shifts for the residues depended on the identity of neighbors or next nearest neighbors. Additional insights into the dynamics of these RNA constructs were gained by molecular dynamics simulations and a discrete path sampling method. Results indicate that the global structures of the RNA are A-form and that the loop regions are dynamic. The results will be useful for understanding the dynamic trajectory of these RNA repeats but also may aid in the development of therapeutics.

Myotonic Dystrophy type 1 (DM1) is caused by an expansion of CUG repeats in DMPK mRNAs. This mutation affects alternative splicing through misregulation of RNA-binding proteins. Amongst pre-mRNAs that are mis-spliced, several code for proteins involved in calcium homeostasis suggesting that calcium-handling and signaling are perturbed in DM1. Here, we analyzed expression of such proteins in DM1 mouse muscle. We found that the levels of several sarcoplasmic reticulum proteins (SERCA1, sarcolipin and calsequestrin) are altered, likely contributing to an imbalance in calcium homeostasis. We also observed that calcineurin (CnA) signaling is hyperactivated in DM1 muscle. Indeed, CnA expression and phosphatase activity are both markedly increased in DM1 muscle. Coherent with this, we found that activators of the CnA pathway (MLP, FHL1) are also elevated. Consequently, NFATc1 expression is increased in DM1 muscle and becomes relocalized to myonuclei, together with an up-regulation of its transcriptional targets (RCAN1.4 and myoglobin). Accordingly, DM1 mouse muscles display an increase in oxidative metabolism and fiber hypertrophy. To determine the functional consequences of this CnA hyperactivation, we administered cyclosporine A, an inhibitor of CnA, to DM1 mice. Muscles of treated DM1 mice showed an increase in CUGBP1 levels, and an exacerbation of key alternative splicing events associated with DM1. Finally, inhibition of CnA in cultured human DM1 myoblasts also resulted in a splicing exacerbation of the insulin receptor. Together, these findings show for the first time that calcium-CnA signaling is hyperactivated in DM1 muscle and that such hyperactivation represents a beneficial compensatory adaptation to the disease., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Myotonic dystrophy type 1 (DM1) is caused by an expansion of CTG repeats in the 3' untranslated region (UTR) of the dystrophia myotonia protein kinase (DMPK) gene. Cognitive impairment associated with structural change in the brain is prevalent in DM1. How this histopathological abnormality during disease progression develops remains elusive. Nuclear accumulation of mutant DMPK mRNA containing expanded CUG RNA disrupting the cytoplasmic and nuclear activities of muscleblind-like (MBNL) protein has been implicated in DM1 neural pathogenesis. The association between MBNL dysfunction and morphological changes has not been investigated. We generated a mouse model for postnatal expression of expanded CUG RNA in the brain that recapitulates the features of the DM1 brain, including the formation of nuclear RNA and MBNL foci, learning disability, brain atrophy and misregulated alternative splicing. Characterization of the pathological abnormalities by a time-course study revealed that hippocampus-related learning and synaptic potentiation were impaired before structural changes in the brain, followed by brain atrophy associated with progressive reduction of axon and dendrite integrity. Moreover, cytoplasmic MBNL1 distribution on dendrites decreased before dendrite degeneration, whereas reduced MBNL2 expression and altered MBNL-regulated alternative splicing was evident after degeneration. These results suggest that the expression of expanded CUG RNA in the DM1 brain results in neurodegenerative processes, with reduced cytoplasmic MBNL1 as an early event response to expanded CUG RNA., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Myotonic dystrophy type 1 (DM1) is a rare multisystemic disorder associated with an expansion of CUG repeats in mutant DMPK (dystrophia myotonica protein kinase) transcripts; the main effect of these expansions is the induction of pre-mRNA splicing defects by sequestering muscleblind-like family proteins (e.g. MBNL1). Disruption of the CUG repeats and the MBNL1 protein complex has been established as the best therapeutic approach for DM1, hence two main strategies have been proposed: targeted degradation of mutant DMPK transcripts and the development of CUG-binding molecules that prevent MBNL1 sequestration. Herein, suitable CUG-binding small molecules were selected using in silico approaches such as scaffold analysis, similarity searching, and druggability analysis. We used polarization assays to confirm the CUG repeat binding in vitro for a number of candidate compounds, and went on to evaluate the biological activity of the two with the strongest affinity for CUG repeats (which we refer to as compounds 1-2 and 2-5) in DM1 mutant cells and Drosophila DM1 models with an impaired locomotion phenotype. In particular, 1-2 and 2-5 enhanced the levels of free MBNL1 in patient-derived myoblasts in vitro and greatly improved DM1 fly locomotion in climbing assays. This work provides new computational approaches for rational large-scale virtual screens of molecules that selectively recognize CUG structures. Moreover, it contributes valuable knowledge regarding two compounds with desirable biological activity in DM1 models.