High-intensity interval training in the form of isometric contraction improves fatigue resistance in dystrophin-deficient muscle.

Duchenne muscular dystrophy is a genetic muscle-wasting disorder characterized by progressive muscle weakness and easy fatigability. Here we examined whether high-intensity interval training (HIIT) in the form of isometric contraction improves fatigue resistance in skeletal muscle from dystrophin-deficient mdx52 mice. Isometric HIIT was performed on plantar flexor muscles in vivo with supramaximal electrical stimulation every other day for 4 weeks (a total of 15 sessions). In the non-trained contralateral gastrocnemius muscle from mdx52 mice, the decreased fatigue resistance was associated with a reduction in the amount of peroxisome proliferator-activated receptor γ coactivator 1-α, citrate synthase activity, mitochondrial respiratory complex II, LC3B-II/I ratio, and mitophagy-related gene expression (i.e. Pink1, parkin, Bnip3 and Bcl2l13) as well as an increase in the phosphorylation levels of Src Tyr416 and Akt Ser473, the amount of p62, and the percentage of Evans Blue dye-positive area. Isometric HIIT restored all these alterations and markedly improved fatigue resistance in mdx52 muscles. Moreover, an acute bout of HIIT increased the phosphorylation levels of AMP-activated protein kinase (AMPK) Thr172, acetyl CoA carboxylase Ser79, unc-51-like autophagy activating kinase 1 (Ulk1) Ser555, and dynamin-related protein 1 (Drp1) Ser616 in mdx52 muscles. Thus, our data show that HIIT with isometric contractions significantly mitigates histological signs of pathology and improves fatigue resistance in dystrophin-deficient muscles. These beneficial effects can be explained by the restoration of mitochondrial function via AMPK-dependent induction of the mitophagy programme and de novo mitochondrial biogenesis. KEY POINTS: Skeletal muscle fatigue is often associated with Duchenne muscular dystrophy (DMD) and leads to an inability to perform daily tasks, profoundly decreasing quality of life. We examined the effect of high-intensity interval training (HIIT) in the form of isometric contraction on fatigue resistance in skeletal muscle from the mdx52 mouse model of DMD. Isometric HIIT counteracted the reduced fatigue resistance as well as dystrophic changes in skeletal muscle of mdx52 mice. This beneficial effect could be explained by the restoration of mitochondrial function via AMP-activated protein kinase-dependent mitochondrial biogenesis and the induction of the mitophagy programme in the dystrophic muscles.

Pharmacological activation of SERCA ameliorates dystrophic phenotypes in dystrophin-deficient mdx mice.

Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder characterized by progressive muscular weakness because of the loss of dystrophin. Extracellular Ca2+ flows into the cytoplasm through membrane tears in dystrophin-deficient myofibers, which leads to muscle contracture and necrosis. Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) takes up cytosolic Ca2+ into the sarcoplasmic reticulum, but its activity is decreased in dystrophic muscle. Here, we show that an allosteric SERCA activator, CDN1163, ameliorates dystrophic phenotypes in dystrophin-deficient mdx mice. The administration of CDN1163 prevented exercise-induced muscular damage and restored mitochondrial function. In addition, treatment with CDN1163 for 7 weeks enhanced muscular strength and reduced muscular degeneration and fibrosis in mdx mice. Our findings provide preclinical proof-of-concept evidence that pharmacological activation of SERCA could be a promising therapeutic strategy for DMD. Moreover, CDN1163 improved muscular strength surprisingly in wild-type mice, which may pave the new way for the treatment of muscular dysfunction.

eSkip-Finder: a machine learning-based web application and database to identify the optimal sequences of antisense oligonucleotides for exon skipping.

Exon skipping using antisense oligonucleotides (ASOs) has recently proven to be a powerful tool for mRNA splicing modulation. Several exon-skipping ASOs have been approved to treat genetic diseases worldwide. However, a significant challenge is the difficulty in selecting an optimal sequence for exon skipping. The efficacy of ASOs is often unpredictable, because of the numerous factors involved in exon skipping. To address this gap, we have developed a computational method using machine-learning algorithms that factors in many parameters as well as experimental data to design highly effective ASOs for exon skipping. eSkip-Finder (https://eskip-finder.org) is the first web-based resource for helping researchers identify effective exon skipping ASOs. eSkip-Finder features two sections: (i) a predictor of the exon skipping efficacy of novel ASOs and (ii) a database of exon skipping ASOs. The predictor facilitates rapid analysis of a given set of exon/intron sequences and ASO lengths to identify effective ASOs for exon skipping based on a machine learning model trained by experimental data. We confirmed that predictions correlated well with in vitro skipping efficacy of sequences that were not included in the training data. The database enables users to search for ASOs using queries such as gene name, species, and exon number.

Establishment of a Triple Quadrupole HPLC-MS Quantitation Method for Dystrophin Protein in Mouse and Human Skeletal Muscle.

Duchenne muscular dystrophy (DMD) is the most common type of neuromuscular disease caused by mutations in the DMD gene encoding dystrophin protein. To quantitively assess human dystrophin protein in muscle biopsy samples, it is imperative to consistently detect as low as 0.003% of the dystrophin protein relative to the total muscle protein content. The quantitation of dystrophin protein has traditionally been conducted using semiquantitative immunoblotting or immunohistochemistry; however, there is a growing need to establish a more precise quantitative method by employing liquid chromatography-mass spectrometry (LC-MS) to measure dystrophin protein. In this study, a novel quantification method was established using a mouse experiment platform applied to the clinical quantification of human dystrophin protein. The method using a spike-in approach with a triple quadrupole LC-MS quantitated the amount of dystrophin in wild-type and human DMD transgenic mice but not in DMD-null mice. In conclusion, we established a quantitating method of dystrophin using HPLC-LC-MS with a novel spike-in approach. These results indicate that our methodology could be applied to several LC-MS devices to enable the accurate measurement of dystrophin protein in patients with DMD.

Caveolin-3 regulates the activity of Ca2+/calmodulin-dependent protein kinase II in C2C12 cells.

Caveolins, encoded by the Cav gene family, are the main components of caveolae. Caveolin-3 (Cav3) is specifically expressed in muscle cells. Mutations in Cav3 are responsible for a group of muscle diseases called caveolinopathies, and Cav3 deficiency is associated with sarcolemmal membrane alterations, disorganization of T-tubules, and disruption of specific cell-signaling pathways. However, Cav3 overexpression increases the number of sarcolemmal caveolae and muscular dystrophy-like regenerating muscle fibers with central nuclei, suggesting that the alteration of Cav3 expression levels or localization influences muscle cell functions. Here, we used mouse C2C12 myoblasts in which Cav3 expression was suppressed with short hairpin RNA and found that Cav3 suppression impaired myotube differentiation without affecting the expression of MyoD and Myog. We also observed an increase of intracellular Ca2+ levels, total calpain activity, and Ca2+-dependent calmodulin kinase II (CaMKII) levels in Cav3-depleted myoblasts. Importantly, those phenotypes due to Cav3 suppression were caused by the ryanodine receptor activation. Furthermore, pharmacological inhibition of CaMKII rescued the impairment of myoblast differentiation due to Cav3 knockdown. Our results suggest that Cav3 regulates intracellular Ca2+ concentrations by modulating ryanodine receptor activity in muscle cells and that CaMKII suppression in muscle could be a novel therapy for caveolinopathies.

Dissociation of SH3 and cysteine-rich domain 3 and junctophilin 1 from dihydropyridine receptor in dystrophin-deficient muscles.

The disruption of excitation-contraction (EC) coupling and subsequent reduction in Ca2+ release from the sarcoplasmic reticulum (SR) have been shown to account for muscle weakness seen in patients with Duchenne muscular dystrophy (DMD). Here, we examined the mechanisms underlying EC uncoupling in skeletal muscles from mdx52 and DMD-null/NSG mice, animal models for DMD, focusing on the SH3 and cysteine-rich domain 3 (STAC3) and junctophilin 1 (JP1), which link the dihydropyridine receptor (DHPR) in the transverse tubule and the ryanodine receptor 1 in the SR. The isometric plantarflexion torque normalized to muscle weight of whole plantar flexor muscles was depressed in mdx52 and DMD-null/NSG mice compared with their control mice. This was accompanied by increased autolysis of calpain-1, decreased levels of STAC3 and JP1 content, and dissociation of STAC3 and JP1 from DHPR-α1s in gastrocnemius muscles. Moreover, in vitro mechanistic experiments demonstrated that STAC3 and JP1 underwent Ca2+-dependent proteolysis that was less pronounced in dystrophin-deficient muscles where calpastatin, the endogenous calpain inhibitor, was upregulated. Eccentric contractions further enhanced autolysis of calpain-1 and proteolysis of STAC3 and JP1 that were associated with severe torque depression in gastrocnemius muscles from DMD-null/NSG mice. These data suggest that Ca2+-dependent proteolysis of STAC3 and JP1 may be an essential factor causing muscle weakness due to EC coupling failure in dystrophin-deficient muscles.NEW & NOTEWORTHY The mechanisms underlying the disruption of excitation-contraction (EC) coupling in dystrophin-deficient muscles are not well understood. Here, using animal models for Duchenne muscular dystrophies (DMD), we show a Ca2+-dependent protease (calpain-1)-mediated proteolysis of SH3 and cysteine-rich domain 3 (STAC3) and junctophilin 1 (JP1), essential EC coupling proteins, in dystrophin-deficient muscle, and highlighting the dissociation of STAC3 and JP1 from dihydropyridine receptor as a causative factor in EC uncoupling of dystrophic muscles.

Mutation-independent Proteomic Signatures of Pathological Progression in Murine Models of Duchenne Muscular Dystrophy.

The absence of the dystrophin protein in Duchenne muscular dystrophy (DMD) results in myofiber fragility and a plethora of downstream secondary pathologies. Although a variety of experimental therapies are in development, achieving effective treatments for DMD remains exceptionally challenging, not least because the pathological consequences of dystrophin loss are incompletely understood. Here we have performed proteome profiling in tibialis anterior muscles from two murine DMD models (mdx and mdx52) at three ages (8, 16, and 80 weeks of age), all n = 3. High-resolution isoelectric focusing liquid chromatography-tandem MS (HiRIEF-LC-MS/MS) was used to quantify the expression of 4974 proteins across all 27 samples. The two dystrophic models were found to be highly similar, whereas multiple proteins were differentially expressed relative to WT (C57BL/6) controls at each age. Furthermore, 1795 proteins were differentially expressed when samples were pooled across ages and dystrophic strains. These included numerous proteins associated with the extracellular matrix and muscle function that have not been reported previously. Pathway analysis revealed multiple perturbed pathways and predicted upstream regulators, which together are indicative of cross-talk between inflammatory, metabolic, and muscle growth pathways (e.g. TNF, INFγ, NF-κB, SIRT1, AMPK, PGC-1α, PPARs, ILK, and AKT/PI3K). Upregulation of CAV3, MVP and PAK1 protein expression was validated in dystrophic muscle by Western blot. Furthermore, MVP was upregulated during, but not required for, the differentiation of C2C12 myoblasts suggesting that this protein may affect muscle regeneration. This study provides novel insights into mutation-independent proteomic signatures characteristic of the dystrophic phenotype and its progression with aging.

The nSMase2/Smpd3 gene modulates the severity of muscular dystrophy and the emotional stress response in mdx mice.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a progressive, degenerative muscular disorder and cognitive dysfunction caused by mutations in the dystrophin gene. It is characterized by excess inflammatory responses in the muscle and repeated degeneration and regeneration cycles. Neutral sphingomyelinase 2/sphingomyelin phosphodiesterase 3 (nSMase2/Smpd3) hydrolyzes sphingomyelin in lipid rafts. This protein thus modulates inflammatory responses, cell survival or apoptosis pathways, and the secretion of extracellular vesicles in a Ca2+-dependent manner. However, its roles in dystrophic pathology have not yet been clarified. METHODS: To investigate the effects of the loss of nSMase2/Smpd3 on dystrophic muscles and its role in the abnormal behavior observed in DMD patients, we generated mdx mice lacking the nSMase2/Smpd3 gene (mdx:Smpd3 double knockout [DKO] mice). RESULTS: Young mdx:Smpd3 DKO mice exhibited reduced muscular degeneration and decreased inflammation responses, but later on they showed exacerbated muscular necrosis. In addition, the abnormal stress response displayed by mdx mice was improved in the mdx:Smpd3 DKO mice, with the recovery of brain-derived neurotrophic factor (Bdnf) expression in the hippocampus. CONCLUSIONS: nSMase2/Smpd3-modulated lipid raft integrity is a potential therapeutic target for DMD.

Severe cardiac involvement with preserved truncated dystrophin expression in Becker muscular dystrophy by +1G>A DMD splice-site mutation: a case report.

Becker muscular dystrophy (BMD) is caused by specific mutations in the DMD gene that causes progressive muscle weakness and primarily affects skeletal and cardiac muscle. Although cardiac involvement is a significant cause of mortality in BMD, the genetic-phenotype correlation for skeletal and cardiac muscles has not been elucidated. Here, we described a 39-year-old man with BMD, who presented with subtle skeletal muscle weakness in the right leg in his 20s and underwent left ventricular restoration for severe dilated cardiomyopathy at the age of 29. He had difficulty climbing stairs after the age of 35. Neither duplication nor deletion of exons was detected by multiplex ligation-dependent probe amplification. A hemizygous c.264 + 1G>A mutation in intron 4 of the DMD was identified by next-generation sequencing. Furthermore, exon 4 skipping of the DMD was confirmed in both skeletal and cardiac muscles evaluated by reverse transcriptase PCR. Endomyocardial and skeletal muscle biopsies revealed dystrophic pathology characterized by muscle fiber atrophy and hypertrophy with a mild degree of interstitial fibrosis. Interestingly, dystrophin immunohistochemistry demonstrated patchy and faint staining of the skeletal muscle membranes but almost normal staining of the cardiac muscle membranes. Western blot analysis revealed a decreased amount of truncated dystrophin in skeletal muscle but surprisingly almost normal amount in cardiac muscle. This case indicates that BMD patients may have severe cardiac dysfunction despite preserved cardiac truncated dystrophin expression.

Autoimmune response and its long-term consequences after exon-skipping therapy in a Duchenne muscular dystrophy mouse model.

The progress of antisense-based therapies using first generation Morpholino oligonucleotides for Duchenne muscular dystrophy (DMD) is expected to partially restore dystrophin expression and may prolong the lifespan of DMD patients. In a recent issue of The Journal of Pathology, a sophisticated study by Vila et al used a dystrophic mouse model of DMD to demonstrate that Morpholino-induced exon skipping induced dystrophin expression in skeletal muscle and stimulated cell mediated and humoral responses to dystrophin. The study highlights the need to further investigate the autoimmune response against de novo synthesised truncated dystrophin protein and its long-term consequences after exon-skipping therapy for DMD. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Exons 45-55 Skipping Using Mutation-Tailored Cocktails of Antisense Morpholinos in the DMD Gene.

Mutations in the dystrophin (DMD) gene and consequent loss of dystrophin cause Duchenne muscular dystrophy (DMD). A promising therapy for DMD, single-exon skipping using antisense phosphorodiamidate morpholino oligomers (PMOs), currently confronts major issues in that an antisense drug induces the production of functionally undefined dystrophin and may not be similarly efficacious among patients with different mutations. Accordingly, the applicability of this approach is limited to out-of-frame mutations. Here, using an exon-skipping efficiency predictive tool, we designed three different PMO cocktail sets for exons 45-55 skipping aiming to produce a dystrophin variant with preserved functionality as seen in milder or asymptomatic individuals with an in-frame exons 45-55 deletion. Of them, the most effective set was composed of select PMOs that each efficiently skips an assigned exon in cell-based screening. These combinational PMOs fitted to different deletions of immortalized DMD patient muscle cells significantly induced exons 45-55 skipping with removing 3, 8, or 10 exons and dystrophin restoration as represented by western blotting. In vivo skipping of the maximum 11 human DMD exons was confirmed in humanized mice. The finding indicates that our PMO set can be used to create mutation-tailored cocktails for exons 45-55 skipping and treat over 65% of DMD patients carrying out-of-frame or in-frame deletions.

Efficacy of Multi-exon Skipping Treatment in Duchenne Muscular Dystrophy Dog Model Neonates.

Duchenne muscular dystrophy (DMD) is caused by mutations in DMD, which codes for dystrophin. Because the progressive and irreversible degeneration of muscle occurs from childhood, earlier therapy is required to prevent dystrophic progression. Exon skipping by antisense oligonucleotides called phosphorodiamidate morpholino oligomers (PMOs), which restores the DMD reading frame and dystrophin expression, is a promising candidate for use in neonatal patients, yet the potential remains unclear. Here, we investigate the systemic efficacy and safety of early exon skipping in dystrophic dog neonates. Intravenous treatment of canine X-linked muscular dystrophy in Japan dogs with a 4-PMO cocktail resulted in ∼3%-27% in-frame exon 6-9 skipping and dystrophin restoration across skeletal muscles up to 14% of healthy levels. Histopathology was ameliorated with the reduction of fibrosis and/or necrosis area and centrally nucleated fibers, significantly in the diaphragm. Treatment induced cardiac multi-exon skipping, though dystrophin rescue was not detected. Functionally, treatment led to significant improvement in the standing test. Toxicity was not observed from blood tests. This is the first study to demonstrate successful multi-exon skipping treatment and significant functional improvement in dystrophic dogs. Early treatment was most beneficial for respiratory muscles, with implications for addressing pulmonary malfunction in patients.

Effects of systemic multiexon skipping with peptide-conjugated morpholinos in the heart of a dog model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal genetic disorder caused by an absence of the dystrophin protein in bodywide muscles, including the heart. Cardiomyopathy is a leading cause of death in DMD. Exon skipping via synthetic phosphorodiamidate morpholino oligomers (PMOs) represents one of the most promising therapeutic options, yet PMOs have shown very little efficacy in cardiac muscle. To increase therapeutic potency in cardiac muscle, we tested a next-generation morpholino: arginine-rich, cell-penetrating peptide-conjugated PMOs (PPMOs) in the canine X-linked muscular dystrophy in Japan (CXMDJ) dog model of DMD. A PPMO cocktail designed to skip dystrophin exons 6 and 8 was injected intramuscularly, intracoronarily, or intravenously into CXMDJ dogs. Intravenous injections with PPMOs restored dystrophin expression in the myocardium and cardiac Purkinje fibers, as well as skeletal muscles. Vacuole degeneration of cardiac Purkinje fibers, as seen in DMD patients, was ameliorated in PPMO-treated dogs. Although symptoms and functions in skeletal muscle were not ameliorated by i.v. treatment, electrocardiogram abnormalities (increased Q-amplitude and Q/R ratio) were improved in CXMDJ dogs after intracoronary or i.v. administration. No obvious evidence of toxicity was found in blood tests throughout the monitoring period of one or four systemic treatments with the PPMO cocktail (12 mg/kg/injection). The present study reports the rescue of dystrophin expression and recovery of the conduction system in the heart of dystrophic dogs by PPMO-mediated multiexon skipping. We demonstrate that rescued dystrophin expression in the Purkinje fibers leads to the improvement/prevention of cardiac conduction abnormalities in the dystrophic heart.

Age-Dependent Echocardiographic and Pathologic Findings in a Rat Model with Duchenne Muscular Dystrophy Generated by CRISPR/Cas9 Genome Editing.

Duchenne muscular dystrophy (DMD) is X-linked recessive myopathy caused by mutations in the dystrophin gene. Although conventional treatments have improved their prognosis, inevitable progressive cardiomyopathy is still the leading cause of death in patients with DMD. To explore novel therapeutic options, a suitable animal model with heart involvement has been warranted.We have generated a rat model with an out-of-frame mutation in the dystrophin gene using CRISPR/Cas9 genome editing (DMD rats). The aim of this study was to evaluate their cardiac functions and pathologies to provide baseline data for future experiments developing treatment options for DMD.In comparison with age-matched wild rats, 6-month-old DMD rats showed no significant differences by echocardiographic evaluations. However, 10-month-old DMD rats showed significant deterioration in left ventricular (LV) fractional shortening (P = 0.024), and in tissue Doppler peak systolic velocity (Sa) at the LV lateral wall (P = 0.041) as well as at the right ventricular (RV) free-wall (P = 0.004). These functional findings were consistent with the fibrotic distributions by histological analysis.Although the cardiac phenotype was milder than anticipated, DMD rats showed similar distributions and progression of heart involvement to those of patients with DMD. This animal may be a useful model with which to develop effective drugs and to understand the underlying mechanisms of progressive heart failure in patients with DMD.

Amelioration of intracellular Ca2+ regulation by exon-45 skipping in Duchenne muscular dystrophy-induced pluripotent stem cell-derived cardiomyocytes.

Duchenne muscular dystrophy (DMD) is a devastating muscle disorder caused by frameshift mutations in the DMD gene. DMD involves cardiac muscle, and the presence of ventricular arrhythmias or congestive failure is critical for prognosis. Several novel therapeutic approaches are being evaluated in ongoing clinical trials. Among them, exon-skipping therapy to correct frameshift mutations with antisense oligonucleotides is promising; however, their therapeutic efficacies on cardiac muscle in vivo remain unknown. In this study, we established induced-pluripotent stem cells (iPSCs) from T cells from a DMD patient carrying a DMD-exon 46-55 deletion, differentiated the iPSCs into cardiomyocytes, and treated them with phosphorodiamidate morpholino oligomers. The efficiency of exon-45 skipping increased in a dose-dependent manner and enabled restoration of the DMD gene product, dystrophin. Further, Ca2+-imaging analysis showed a decreased number of arrhythmic cells and improved transient Ca2+ signaling after exon skipping. Thus, exon-45 skipping may be effective for cardiac involvement in DMD patients harboring the DMD-exon 46-55 deletion.

Truncated dystrophin ameliorates the dystrophic phenotype of mdx mice by reducing sarcolipin-mediated SERCA inhibition.

Duchenne muscular dystrophy (DMD) and the less severe Becker muscular dystrophy (BMD) are due to mutations in the DMD gene. Previous reports show that in-frame deletion of exons 45-55 produces an internally shorted, but functional, dystrophin protein resulting in a very mild BMD phenotype. In order to elucidate the molecular mechanism leading to this phenotype, we generated exon 45-55 deleted dystrophin transgenic/mdx (Tg/mdx) mice. Muscular function of Tg/mdx mice was restored close to that of wild type (WT) mice but the localization of the neuronal type of nitric oxide synthase was changed from the sarcolemma to the cytosol. This led to hyper-nitrosylation of the ryanodine receptor 1 causing increased Ca2+ release from the sarcoplasmic reticulum. On the other hand, Ca2+ reuptake by the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) was restored to the level of WT mice, suggesting that the Ca2+ dysregulation had been compensated by SERCA activation. In line with this, expression of sarcolipin (SLN), a SERCA-inhibitory peptide, was upregulated in mdx mice, but strongly reduced in Tg/mdx mice. Furthermore, knockdown of SLN ameliorated the cytosolic Ca2+ homeostasis and the dystrophic phenotype in mdx mice. These findings suggest that SLN may be a novel target for DMD therapy.

C9orf72 and RAB7L1 regulate vesicle trafficking in amyotrophic lateral sclerosis and frontotemporal dementia.

A non-coding hexanucleotide repeat expansion in intron 1 of the C9orf72 gene is the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), however, the precise molecular mechanism by which the C9orf72 hexanucleotide repeat expansion directs C9ALS/FTD pathogenesis remains unclear. Here, we report a novel disease mechanism arising due to the interaction of C9ORF72 with the RAB7L1 GTPase to regulate vesicle trafficking. Endogenous interaction between C9ORF72 and RAB7L1 was confirmed in human SH-SY5Y neuroblastoma cells. The C9orf72 hexanucleotide repeat expansion led to haploinsufficiency resulting in severely defective intracellular and extracellular vesicle trafficking and a dysfunctional trans-Golgi network phenotype in patient-derived fibroblasts and induced pluripotent stem cell-derived motor neurons. Genetic ablation of RAB7L1or C9orf72 in SH-SY5Y cells recapitulated the findings in C9ALS/FTD fibroblasts and induced pluripotent stem cell neurons. When C9ORF72 was overexpressed or antisense oligonucleotides were targeted to the C9orf72 hexanucleotide repeat expansion to upregulate normal variant 1 transcript levels, the defective vesicle trafficking and dysfunctional trans-Golgi network phenotypes were reversed, suggesting that both loss- and gain-of-function mechanisms play a role in disease pathogenesis. In conclusion, we have identified a novel mechanism for C9ALS/FTD pathogenesis highlighting the molecular regulation of intracellular and extracellular vesicle trafficking as an important pathway in C9ALS/FTD pathogenesis.

Quantitative Antisense Screening and Optimization for Exon 51 Skipping in Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD), the most common lethal genetic disorder, is caused by mutations in the dystrophin (DMD) gene. Exon skipping is a therapeutic approach that uses antisense oligonucleotides (AOs) to modulate splicing and restore the reading frame, leading to truncated, yet functional protein expression. In 2016, the US Food and Drug Administration (FDA) conditionally approved the first phosphorodiamidate morpholino oligomer (morpholino)-based AO drug, eteplirsen, developed for DMD exon 51 skipping. Eteplirsen remains controversial with insufficient evidence of its therapeutic effect in patients. We recently developed an in silico tool to design antisense morpholino sequences for exon skipping. Here, we designed morpholino AOs targeting DMD exon 51 using the in silico tool and quantitatively evaluated the effects in immortalized DMD muscle cells in vitro. To our surprise, most of the newly designed morpholinos induced exon 51 skipping more efficiently compared with the eteplirsen sequence. The efficacy of exon 51 skipping and rescue of dystrophin protein expression were increased by up to more than 12-fold and 7-fold, respectively, compared with the eteplirsen sequence. Significant in vivo efficacy of the most effective morpholino, determined in vitro, was confirmed in mice carrying the human DMD gene. These findings underscore the importance of AO sequence optimization for exon skipping.

Serum Osteopontin as a Novel Biomarker for Muscle Regeneration in Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy is a lethal X-linked muscle disorder. We have already reported that osteopontin (OPN), an inflammatory cytokine and myogenic factor, is expressed in the early dystrophic phase in canine X-linked muscular dystrophy in Japan, a dystrophic dog model. To further explore the possibility of OPN as a new biomarker for disease activity in Duchenne muscular dystrophy, we monitored serum OPN levels in dystrophic and wild-type dogs at different ages and compared the levels to other serum markers, such as serum creatine kinase, matrix metalloproteinase-9, and tissue inhibitor of metalloproteinase-1. Serum OPN levels in the dystrophic dogs were significantly elevated compared with those in wild-type dogs before and 1 hour after a cesarean section birth and at the age of 3 months. The serum OPN level was significantly correlated with the phenotypic severity of dystrophic dogs at the period corresponding to the onset of muscle weakness, whereas other serum markers including creatine kinase were not. Immunohistologically, OPN was up-regulated in infiltrating macrophages and developmental myosin heavy chain-positive regenerating muscle fibers in the dystrophic dogs, whereas serum OPN was highly elevated. OPN expression was also observed during the synergic muscle regeneration process induced by cardiotoxin injection. In conclusion, OPN is a promising biomarker for muscle regeneration in dystrophic dogs and can be applicable to boys with Duchenne muscular dystrophy.

Deletion of exons 3-9 encompassing a mutational hot spot in the DMD gene presents an asymptomatic phenotype, indicating a target region for multiexon skipping therapy.

Few cases of dystrophinopathy show an asymptomatic phenotype with mutations in the 5' (exons 3-7) hot spot in the Duchenne muscular dystrophy (DMD) gene. Our patient showed increased serum creatine kinase levels at 12 years of age. A muscle biopsy at 15 years of age led to a diagnosis of Becker muscular dystrophy. The patient showed a slight decrease in cardiac function at the age of 21 years and was administered a ß-blocker, but there was no muscle involvement even at the age of 27 years. A deletion of exons 3-9 encompassing a mutational hot spot in the DMD gene was detected, and dystrophin protein expression was ∼15% that of control level. We propose that in-frame deletion of exons 3-9 may produce a functional protein, and that multiexon skipping therapy targeting these exons may be feasible for severe dystrophic patients with a mutation in the 5' hot spot of the DMD gene.