Therapeutic potential of oleic acid supplementation in myotonic dystrophy muscle cell models.

BACKGROUND: We recently reported that upregulation of Musashi 2 (MSI2) protein in the rare neuromuscular disease myotonic dystrophy type 1 contributes to the hyperactivation of the muscle catabolic processes autophagy and UPS through a reduction in miR-7 levels. Because oleic acid (OA) is a known allosteric regulator of MSI2 activity in the biogenesis of miR-7, here we sought to evaluate endogenous levels of this fatty acid and its therapeutic potential in rescuing cell differentiation phenotypes in vitro. In this work, four muscle cell lines derived from DM1 patients were treated with OA for 24 h, and autophagy and muscle differentiation parameters were analyzed. RESULTS: We demonstrate a reduction of OA levels in different cell models of the disease. OA supplementation rescued disease-related phenotypes such as fusion index, myotube diameter, and repressed autophagy. This involved inhibiting MSI2 regulation of direct molecular target miR-7 since OA isoschizomer, elaidic acid (EA) could not cause the same rescues. Reduction of OA levels seems to stem from impaired biogenesis since levels of the enzyme stearoyl-CoA desaturase 1 (SCD1), responsible for converting stearic acid to oleic acid, are decreased in DM1 and correlate with OA amounts. CONCLUSIONS: For the first time in DM1, we describe a fatty acid metabolism impairment that originated, at least in part, from a decrease in SCD1. Because OA allosterically inhibits MSI2 binding to molecular targets, reduced OA levels synergize with the overexpression of MSI2 and contribute to the MSI2 > miR-7 > autophagy axis that we proposed to explain the muscle atrophy phenotype.

Neuroprotective properties of queen bee acid by autophagy induction.

Autophagy is a conserved intracellular catabolic pathway that removes cytoplasmic components to contribute to neuronal homeostasis. Accumulating evidence has increasingly shown that the induction of autophagy improves neuronal health and extends longevity in several animal models. Therefore, there is a great interest in the identification of effective autophagy enhancers with potential nutraceutical or pharmaceutical properties to ameliorate age-related diseases, such as neurodegenerative disorders, and/or promote longevity. Queen bee acid (QBA, 10-hydroxy-2-decenoic acid) is the major fatty acid component of, and is found exclusively in, royal jelly, which has beneficial properties for human health. It is reported that QBA has antitumor, anti-inflammatory, and antibacterial activities and promotes neurogenesis and neuronal health; however, the mechanism by which QBA exerts these effects has not been fully elucidated. The present study investigated the role of the autophagic process in the protective effect of QBA. We found that QBA is a novel autophagy inducer that triggers autophagy in various neuronal cell lines and mouse and fly models. The beclin-1 (BECN1) and mTOR pathways participate in the regulation of QBA-induced autophagy. Moreover, our results showed that QBA stimulates sirtuin 1 (SIRT1), which promotes autophagy by the deacetylation of critical ATG proteins. Finally, QBA-mediated autophagy promotes neuroprotection in Parkinson's disease in vitro and in a mouse model and extends the lifespan of Drosophila melanogaster. This study provides detailed evidences showing that autophagy induction plays a critical role in the beneficial health effects of QBA.

Moxifloxacin rescues SMA phenotypes in patient-derived cells and animal model.

Spinal muscular atrophy (SMA) is a genetic disease resulting in the loss of α-motoneurons followed by muscle atrophy. It is caused by knock-out mutations in the survival of motor neuron 1 (SMN1) gene, which has an unaffected, but due to preferential exon 7 skipping, only partially functional human-specific SMN2 copy. We previously described a Drosophila-based screening of FDA-approved drugs that led us to discover moxifloxacin. We showed its positive effect on the SMN2 exon 7 splicing in SMA patient-derived skin cells and its ability to increase the SMN protein level. Here, we focus on moxifloxacin's therapeutic potential in additional SMA cellular and animal models. We demonstrate that moxifloxacin rescues the SMA-related molecular and phenotypical defects in muscle cells and motoneurons by improving the SMN2 splicing. The consequent increase of SMN levels was higher than in case of risdiplam, a potent exon 7 splicing modifier, and exceeded the threshold necessary for a survival improvement. We also demonstrate that daily subcutaneous injections of moxifloxacin in a severe SMA murine model reduces its characteristic neuroinflammation and increases the SMN levels in various tissues, leading to improved motor skills and extended lifespan. We show that moxifloxacin, originally used as an antibiotic, can be potentially repositioned for the SMA treatment.

Natural Compound Boldine Lessens Myotonic Dystrophy Type 1 Phenotypes in DM1 Drosophila Models, Patient-Derived Cell Lines, and HSALR Mice.

Myotonic dystrophy type 1 (DM1) is a complex rare disorder characterized by progressive muscle dysfunction, involving weakness, myotonia, and wasting, but also exhibiting additional clinical signs in multiple organs and systems. Central dysregulation, caused by an expansion of a CTG trinucleotide repeat in the DMPK gene's 3' UTR, has led to exploring various therapeutic approaches in recent years, a few of which are currently under clinical trial. However, no effective disease-modifying treatments are available yet. In this study, we demonstrate that treatments with boldine, a natural alkaloid identified in a large-scale Drosophila-based pharmacological screening, was able to modify disease phenotypes in several DM1 models. The most significant effects include consistent reduction in nuclear RNA foci, a dynamic molecular hallmark of the disease, and noteworthy anti-myotonic activity. These results position boldine as an attractive new candidate for therapy development in DM1.

Inhibition of autophagy rescues muscle atrophy in a LGMDD2 Drosophila model.

Limb-girdle muscular dystrophy D2 (LGMDD2) is an ultrarare autosomal dominant myopathy caused by mutation of the normal stop codon of the TNPO3 nuclear importin. The mutant protein carries a 15 amino acid C-terminal extension associated with pathogenicity. Here we report the first animal model of the disease by expressing the human mutant TNPO3 gene in Drosophila musculature or motor neurons and concomitantly silencing the endogenous expression of the fly protein ortholog. A similar genotype expressing wildtype TNPO3 served as a control. Phenotypes characterization revealed that mutant TNPO3 expression targeted at muscles or motor neurons caused LGMDD2-like phenotypes such as muscle degeneration and atrophy, and reduced locomotor ability. Notably, LGMDD2 mutation increase TNPO3 at the transcript and protein level in the Drosophila model Upregulated muscle autophagy observed in LGMDD2 patients was also confirmed in the fly model, in which the anti-autophagic drug chloroquine was able to rescue histologic and functional phenotypes. Overall, we provide a proof of concept of autophagy as a target to treat disease phenotypes and propose a neurogenic component to explain mutant TNPO3 pathogenicity in diseased muscles.

Increased Muscleblind levels by chloroquine treatment improve myotonic dystrophy type 1 phenotypes in in vitro and in vivo models.

Myotonic dystrophy type 1 (DM1) is a life-threatening and chronically debilitating neuromuscular disease caused by the expansion of a CTG trinucleotide repeat in the 3' UTR of the DMPK gene. The mutant RNA forms insoluble structures capable of sequestering RNA binding proteins of the Muscleblind-like (MBNL) family, which ultimately leads to phenotypes. In this work, we demonstrate that treatment with the antiautophagic drug chloroquine was sufficient to up-regulate MBNL1 and 2 proteins in Drosophila and mouse (HSALR) models and patient-derived myoblasts. Extra Muscleblind was functional at the molecular level and improved splicing events regulated by MBNLs in all disease models. In vivo, chloroquine restored locomotion, rescued average cross-sectional muscle area, and extended median survival in DM1 flies. In HSALR mice, the drug restored muscular strength and histopathology signs and reduced the grade of myotonia. Taken together, these results offer a means to replenish critically low MBNL levels in DM1.

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

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

Drosophila SMN2 minigene reporter model identifies moxifloxacin as a candidate therapy for SMA.

Spinal muscular atrophy is a rare and fatal neuromuscular disorder caused by the loss of alpha motor neurons. The affected individuals have mutated the ubiquitously expressed SMN1 gene resulting in the loss or reduction in the survival motor neuron (SMN) protein levels. However, an almost identical paralog exists in humans: SMN2. Pharmacological activation of SMN2 exon 7 inclusion by small molecules or modified antisense oligonucleotides is a valid approach to treat SMA. Here we describe an in vivo SMN2 minigene reporter system in Drosophila motor neurons that serves as a cost-effective, feasible, and stringent primary screening model for identifying chemicals capable of crossing the conserved Drosophila blood-brain barrier and modulating exon 7 inclusion. The model was used for the screening of 1100 drugs from the Prestwick Chemical Library, resulting in 2.45% hit rate. The most promising candidate drugs were validated in patient-derived fibroblasts where they proved to increase SMN protein levels. Among them, moxifloxacin modulated SMN2 splicing by promoting exon 7 inclusion. The recovery of SMN protein levels was confirmed by increased colocalization of nuclear gems with Cajal Bodies. Thus, a Drosophila-based drug screen allowed the discovery of an FDA-approved small molecule with the potential to become a novel therapy for SMA.

MicroRNA-Based Therapeutic Perspectives in Myotonic Dystrophy.

Myotonic dystrophy involves two types of chronically debilitating rare neuromuscular diseases: type 1 (DM1) and type 2 (DM2). Both share similarities in molecular cause, clinical signs, and symptoms with DM2 patients usually displaying milder phenotypes. It is well documented that key clinical symptoms in DM are associated with a strong mis-regulation of RNA metabolism observed in patient's cells. This mis-regulation is triggered by two leading DM-linked events: the sequestration of Muscleblind-like proteins (MBNL) and the mis-regulation of the CUGBP RNA-Binding Protein Elav-Like Family Member 1 (CELF1) that cause significant alterations to their important functions in RNA processing. It has been suggested that DM1 may be treatable through endogenous modulation of the expression of MBNL and CELF1 proteins. In this study, we analyzed the recent identification of the involvement of microRNA (miRNA) molecules in DM and focus on the modulation of these miRNAs to therapeutically restore normal MBNL or CELF1 function. We also discuss additional prospective miRNA targets, the use of miRNAs as disease biomarkers, and additional promising miRNA-based and miRNA-targeting drug development strategies. This review provides a unifying overview of the dispersed data on miRNA available in the context of DM.

Expanded CTG repeats trigger miRNA alterations in Drosophila that are conserved in myotonic dystrophy type 1 patients.

Myotonic dystrophy type 1 (DM1) is caused by the expansion of CTG repeats in the 3' untranslated region of the DMPK gene. Several missplicing events and transcriptional alterations have been described in DM1 patients. A large number of these defects have been reproduced in animal models expressing CTG repeats alone. Recent studies have also reported miRNA dysregulation in DM1 patients. In this work, a Drosophila model was used to investigate miRNA transcriptome alterations in the muscle, specifically triggered by CTG expansions. Twenty miRNAs were differentially expressed in CTG-expressing flies. Of these, 19 were down-regulated, whereas 1 was up-regulated. This trend was confirmed for those miRNAs conserved between Drosophila and humans (miR-1, miR-7 and miR-10) in muscle biopsies from DM1 patients. Consistently, at least seven target transcripts of these miRNAs were up-regulated in DM1 skeletal muscles. The mechanisms involved in dysregulation of miR-7 included a reduction of its primary precursor both in CTG-expressing flies and in DM1 patients. Additionally, a regulatory role for Muscleblind (Mbl) was also suggested for miR-1 and miR-7, as these miRNAs were down-regulated in flies where Mbl had been silenced. Finally, the physiological relevance of miRNA dysregulation was demonstrated for miR-10, since over-expression of this miRNA in Drosophila extended the lifespan of CTG-expressing flies. Taken together, our results contribute to our understanding of the origin and the role of miRNA alterations in DM1.

The insect nephrocyte is a podocyte-like cell with a filtration slit diaphragm.

The nephron is the basic structural and functional unit of the vertebrate kidney. It is composed of a glomerulus, the site of ultrafiltration, and a renal tubule, along which the filtrate is modified. Although widely regarded as a vertebrate adaptation, 'nephron-like' features can be found in the excretory systems of many invertebrates, raising the possibility that components of the vertebrate excretory system were inherited from their invertebrate ancestors. Here we show that the insect nephrocyte has remarkable anatomical, molecular and functional similarity to the glomerular podocyte, a cell in the vertebrate kidney that forms the main size-selective barrier as blood is ultrafiltered to make urine. In particular, both cell types possess a specialized filtration diaphragm, known as the slit diaphragm in podocytes or the nephrocyte diaphragm in nephrocytes. We find that fly (Drosophila melanogaster) orthologues of the major constituents of the slit diaphragm, including nephrin, NEPH1 (also known as KIRREL), CD2AP, ZO-1 (TJP1) and podocin, are expressed in the nephrocyte and form a complex of interacting proteins that closely mirrors the vertebrate slit diaphragm complex. Furthermore, we find that the nephrocyte diaphragm is completely lost in flies lacking the orthologues of nephrin or NEPH1-a phenotype resembling loss of the slit diaphragm in the absence of either nephrin (as in human congenital nephrotic syndrome of the Finnish type, NPHS1) or NEPH1. These changes markedly impair filtration function in the nephrocyte. The similarities we describe between invertebrate nephrocytes and vertebrate podocytes provide evidence suggesting that the two cell types are evolutionarily related, and establish the nephrocyte as a simple model in which to study podocyte biology and podocyte-associated diseases.

In vivo discovery of a peptide that prevents CUG-RNA hairpin formation and reverses RNA toxicity in myotonic dystrophy models.

Myotonic dystrophy type 1 (DM1) is caused by the expansion of noncoding CTG repeats in the dystrophia myotonica-protein kinase gene. Mutant transcripts form CUG hairpins that sequester RNA-binding factors into nuclear foci, including Muscleblind-like-1 protein (MBNL1), which regulate alternative splicing and gene expression. To identify molecules that target toxic CUG transcripts in vivo, we performed a positional scanning combinatorial peptide library screen using a Drosophila model of DM1. The screen identified a D-amino acid hexapeptide (ABP1) that reduced CUG foci formation and suppressed CUG-induced lethality and muscle degeneration when administered orally. Transgenic expression of natural, L-amino acid ABP1 analogues reduced CUG-induced toxicity in fly eyes and muscles. Furthermore, ABP1 reversed muscle histopathology and splicing misregulation of MBNL1 targets in DM1 model mice. In vitro, ABP1 bound to CUG hairpins and induced a switch to a single-stranded conformation. Our findings demonstrate that ABP1 shows antimyotonic dystrophy activity by targeting the core of CUG toxicity.

Taurine activates the AKT-mTOR axis to restore muscle mass and contractile strength in human 3D in vitro models of steroid myopathy.

Steroid myopathy is a clinically challenging condition exacerbated by prolonged corticosteroid use or adrenal tumors. In this study, we engineered a functional three-dimensional (3D) in vitro skeletal muscle model to investigate steroid myopathy. By subjecting our bioengineered muscle tissues to dexamethasone treatment, we reproduced the molecular and functional aspects of this disease. Dexamethasone caused a substantial reduction in muscle force, myotube diameter and induced fatigue. We observed nuclear translocation of the glucocorticoid receptor (GCR) and activation of the ubiquitin-proteasome system within our model, suggesting their coordinated role in muscle atrophy. We then examined the therapeutic potential of taurine in our 3D model for steroid myopathy. Our findings revealed an upregulation of phosphorylated AKT by taurine, effectively countering the hyperactivation of the ubiquitin-proteasomal pathway. Importantly, we demonstrate that discontinuing corticosteroid treatment was insufficient to restore muscle mass and function. Taurine treatment, when administered concurrently with corticosteroids, notably enhanced contractile strength and protein turnover by upregulating the AKT-mTOR axis. Our model not only identifies a promising therapeutic target, but also suggests combinatorial treatment that may benefit individuals undergoing corticosteroid treatment or those diagnosed with adrenal tumors.

In vivo strategies for drug discovery in myotonic dystrophy disorders.

Myotonic dystrophy (DM) is a complex neuromuscular genetic disease for which there is currently no valid therapy. The recent development of non-mammal animal models opened up the possibility of performing drug discovery in vivo, using as screening readout phenotypes with underlying molecular parallels to the disease. In this review we discuss the state of the art technologies already used in large scale drug screening and provide guidance for further development of novel technologies.

The myotonic dystrophy type 1 drug development pipeline: 2022 edition.

The beginning of the 20th decade has witnessed an increase in drug development programs for myotonic dystrophy type 1 (DM1). We have collected nearly 20 candidate drugs with accomplished preclinical and clinical phases, updating our previous drug development pipeline review with new entries and relevant milestones for pre-existing candidates. Three interventional first-in-human clinical trials got underway with distinct drug classes, namely AOC 1001 and DYNE-101 nucleic acid-based therapies, and the small molecule pitolisant, which joins the race toward market authorization with other repurposed drugs, including tideglusib, metformin, or mexiletine, already in clinical evaluation. Furthermore, newly disclosed promising preclinical data for several additional nucleic-acid therapeutic candidates and a CRISPR-based approach, as well as the advent into the pipeline of novel therapeutic programs, increase the plausibility of success in the demanding task of providing valid treatments to patients with DM1.

Quantitative magnetic resonance imaging assessment of muscle composition in myotonic dystrophy mice.

Myotonic dystrophy type 1 (DM1) is a severe autosomal dominant neuromuscular disease in which the musculoskeletal system contributes substantially to overall mortality and morbidity. DM1 stems from a noncoding CTG trinucleotide repeat expansion in the DMPK gene. The human skeletal actin long repeat (HSALR) mouse model reproduces several aspects of the disease, but the muscle-wasting phenotype of this model has never been characterized in vivo. Herein, we used quantitative MRI to measure the fat and muscle volumes in the leg compartment (LC) of mice. These acquired data were processed to extract relevant parameters such as fat fraction and fat infiltration (fat LC/LC) in HSALR and control (FBV) muscles. These results showed increased fat volume (fat LC) and fat infiltration within the muscle tissue of the leg compartment (muscle LC), in agreement with necropsies, in which fatty clumps were observed, and consistent with previous findings in DM1 patients. Model mice did not reproduce the characteristic impaired fat fraction, widespread fat replacement through the muscles, or reduced muscle volume reported in patients. Taken together, the observed abnormal replacement of skeletal muscle by fat in the HSALR mice indicates that these mice partially reproduced the muscle phenotype observed in humans.

Msi2 enhances muscle dysfunction in a myotonic dystrophy type 1 mouse model.

BACKGROUND: Myotonic dystrophy type 1 (DM1) is a rare neuromuscular disease caused by a CTG repeat expansion in the 3' untranslated region of the DM1 protein kinase gene. Characteristic degenerative muscle symptoms include myotonia, atrophy, and weakness. We previously proposed an MSI2>miR-7>autophagy axis whereby MSI2 overexpression repressed miR-7 biogenesis that subsequently de-repressed muscle catabolism through excessive autophagy. Because the DM1 HSALR mouse model expressing expanded CUG repeats shows weak muscle-wasting phenotypes, we hypothesized that MSI2 overexpression was sufficient to promote muscle dysfunction in vivo. METHODS: By means of recombinant AAV murine Msi2 was overexpressed in neonates HSALR mice skeletal muscle to induce DM1-like phenotypes RESULTS: Sustained overexpression of the murine Msi2 protein in HSALR neonates induced autophagic flux and expression of critical autophagy proteins, increased central nuclei and reduced myofibers area, and weakened muscle strength. Importantly, these changes were independent of Mbnl1, Mbnl2, and Celf1 protein levels, which remained unchanged upon Msi2 overexpression. CONCLUSIONS: Globally, molecular, histological, and functional data from these experiments in the HSALR mouse model confirms the pathological role of Msi2 expression levels as an atrophy-associated component that impacts the characteristic muscle dysfunction symptoms in DM1 patients.

CRISPR-Cas9 editing of a TNPO3 mutation in a muscle cell model of limb-girdle muscular dystrophy type D2.

A single-nucleotide deletion in the stop codon of the nuclear import receptor transportin-3 (TNPO3), also involved in human immunodeficiency virus type 1 (HIV-1) infection, causes the ultrarare autosomal dominant disease limb-girdle muscular dystrophy D2 (LGMDD2) by extending the wild-type protein. Here, we generated a patient-derived in vitro model of LGMDD2 as an immortalized myoblast cell line carrying the TNP O 3 mutation. The cell model reproduced critical molecular alterations seen in patients, such as TNP O 3 overexpression, defects in terminal muscle markers, and autophagy overactivation. Correction of the TNP O 3 mutation via CRISPR-Cas9 editing caused a significant reversion of the pathological phenotypes in edited cells, including a complete absence of the mutant TNPO3 protein, as detected with a polyclonal antibody specific against the abnormal 15-aa peptide. Transcriptomic analyses found that 15% of the transcriptome was differentially expressed in model myotubes. CRISPR-Cas9-corrected cells showed that 44% of the alterations were rescued toward normal levels. MicroRNAs (miRNAs) analyses showed that around 50% of miRNAs with impaired expression because of the disease were recovered on the mutation edition. In summary, this work provides proof of concept of the potential of CRISPR-Cas9-mediated gene editing of TNP O 3 as a therapeutic approach and describes critical reagents in LGMDD2 research.

BlockmiR AONs as Site-Specific Therapeutic MBNL Modulation in Myotonic Dystrophy 2D and 3D Muscle Cells and HSALR Mice.

The symptoms of Myotonic Dystrophy Type 1 (DM1) are multi-systemic and life-threatening. The neuromuscular disorder is rooted in a non-coding CTG microsatellite expansion in the DM1 protein kinase (DMPK) gene that, upon transcription, physically sequesters the Muscleblind-like (MBNL) family of splicing regulator proteins. The high-affinity binding occurring between the proteins and the repetitions disallow MBNL proteins from performing their post-transcriptional splicing regulation leading to downstream molecular effects directly related to disease symptoms such as myotonia and muscle weakness. In this study, we build on previously demonstrated evidence showing that the silencing of miRNA-23b and miRNA-218 can increase MBNL1 protein in DM1 cells and mice. Here, we use blockmiR antisense technology in DM1 muscle cells, 3D mouse-derived muscle tissue, and in vivo mice to block the binding sites of these microRNAs in order to increase MBNL translation into protein without binding to microRNAs. The blockmiRs show therapeutic effects with the rescue of mis-splicing, MBNL subcellular localization, and highly specific transcriptomic expression. The blockmiRs are well tolerated in 3D mouse skeletal tissue inducing no immune response. In vivo, a candidate blockmiR also increases Mbnl1/2 protein and rescues grip strength, splicing, and histological phenotypes.

Peptide-conjugated antimiRs improve myotonic dystrophy type 1 phenotypes by promoting endogenous MBNL1 expression.

Myotonic dystrophy type 1 (DM1) is a rare neuromuscular disease caused by a CTG repeat expansion in the DMPK gene that generates toxic RNA with a myriad of downstream alterations in RNA metabolism. A key consequence is the sequestration of alternative splicing regulatory proteins MBNL1/2 by expanded transcripts in the affected tissues. MBNL1/2 depletion interferes with a developmental alternative splicing switch that causes the expression of fetal isoforms in adults. Boosting the endogenous expression of MBNL proteins by inhibiting the natural translational repressors miR-23b and miR-218 has previously been shown to be a promising therapeutic approach. We designed antimiRs against both miRNAs with a phosphorodiamidate morpholino oligonucleotide (PMO) chemistry conjugated to cell-penetrating peptides (CPPs) to improve delivery to affected tissues. In DM1 cells, CPP-PMOs significantly increased MBNL1 levels. In some candidates, this was achieved using concentrations less than two orders of magnitude below the median toxic concentration, with up to 5.38-fold better therapeutic window than previous antagomiRs. In HSALR mice, intravenous injections of CPP-PMOs improve molecular, histopathological, and functional phenotypes, without signs of toxicity. Our findings place CPP-PMOs as promising antimiR candidates to overcome the treatment delivery challenge in DM1 therapy.