Search

Neurofilament levels are elevated in many neurodegenerative diseases and have shown promise as diagnostic and prognostic biomarkers in Amyotrophic Lateral Sclerosis (ALS), the most common form of Motor Neuron Disease (MND). This study assesses serum neurofilament light (NFL) and neurofilament heavy (NFH) chain concentrations in patients with ALS, other variants of motor neuron disease such as Progressive Muscular Atrophy (PMA) and Primary Lateral Sclerosis (PLS), and a range of other neurological diseases. It aims to evaluate the use of NFL and NFH to differentiate these conditions and for the prognosis of MND disease progression. NFL and NFH levels were quantified using electrochemiluminescence immunoassays (ECLIA). Both were elevated in 47 patients with MND compared to 34 patients with other neurological diseases and 33 healthy controls. NFL was able to differentiate patients with MND from the other groups with a Receiver Operating Characteristic (ROC) curve area under the curve (AUC) of 0.90 (p < 0.001). NFL correlated with the rate of disease progression in MND (rho 0.758, p < 0.001) and with the ALS Functional Rating Scale (rho −0.335, p = 0.021). NFL levels were higher in patients with ALS compared to both PMA (p = 0.032) and PLS (p = 0.012) and were able to distinguish ALS from both PMA and PLS with a ROC curve AUC of 0.767 (p = 0.005). These findings support the use of serum NFL to help diagnose and differentiate types of MND, in addition to providing prognostic information to patients and their families.

Amyotrophic lateral sclerosis (ALS) is a fatal late-onset motor neuron disease characterized by the loss of the upper and lower motor neurons. Our understanding of the molecular basis of ALS pathology remains elusive, complicating the development of efficient treatment. Gene-set analyses of genome-wide data have offered insight into the biological processes and pathways of complex diseases and can suggest new hypotheses regarding causal mechanisms. Our aim in this study was to identify and explore biological pathways and other gene sets having genomic association to ALS. Two cohorts of genomic data from the dbGaP repository were combined: (a) the largest available ALS individual-level genotype dataset (N = 12,319), and (b) a similarly sized control cohort (N = 13,210). Following comprehensive quality control pipelines, imputation and meta-analysis, we assembled a large European descent ALS-control cohort of 9244 ALS cases and 12,795 healthy controls represented by genetic variants of 19,242 genes. Multi-marker analysis of genomic annotation (MAGMA) gene-set analysis was applied to an extensive collection of 31,454 gene sets from the molecular signatures database (MSigDB). Statistically significant associations were observed for gene sets related to immune response, apoptosis, lipid metabolism, neuron differentiation, muscle cell function, synaptic plasticity and development. We also report novel interactions between gene sets, suggestive of mechanistic overlaps. A manual meta-categorization and enrichment mapping approach is used to explore the overlap of gene membership between significant gene sets, revealing a number of shared mechanisms.

BACKGROUND AND PURPOSE This study evaluates the incidence, prevalence and survival trends of motor neurone disease (MND) in Northern Ireland from 2015 to 2019. METHODS A capture-recapture analysis was performed using five independent data sources. Incidence and prevalence rates were standardized to the European Standard Population. Survival outcomes were analysed using Kaplan-Meier curves and Cox regression analysis. RESULTS Amongst 254 total cases of MND, capture-recapture analysis estimated three missing cases (case ascertainment 98.8%). Age standardized incidence of captured cases was 3.12 per 100,000 (2.73, 3.50) and standardized prevalence ranged from 9.45 to 6.49 per 100,000 from 2015 to 2019. Standardized incidence and prevalence rates in 2006 were 1.4 and 3.3 per 100,000 respectively. Of identified cases, 133 (52.4%) were male; 94.5% had amyotrophic lateral sclerosis; median age of onset was 67 years; median time to diagnosis was 12 months (95% confidence interval 11.2, 12.8); survival from diagnosis was 12 months (95% confidence interval 10.6, 15.4); 25 (9.8%) reported a family history of MND or frontotemporal dementia; and a known MND-associated genetic mutation was identified in 7.9% of total cases, of which the most common was C9orf72 (5.7% of all patients). Factors associated with improved survival were younger age at onset, longer time to diagnosis, attendance at regional MND clinic, and initial neurology presentation as outpatient (all p

Background Facioscapulohumeral dystrophy (FSHD) is caused by mutations leading to the aberrant expression of the DUX4 transcription factor in muscles. DUX4 was proposed to induce cell death, but the involvement of different death pathways is still discussed. A possible pro‐apoptotic role of DUX4 was proposed, but as FSHD muscles are characterized by necrosis and inflammatory infiltrates, non‐apoptotic pathways may be also involved. Methods We explored DUX4‐mediated cell death by focusing on the role of one regulated necrosis pathway called necroptosis, which is regulated by RIPK3. We investigated the effect of necroptosis on cell death in vitro and in vivo experiments using RIPK3 inhibitors and a RIPK3‐deficient transgenic mouse model. Results We showed in vitro that DUX4 expression causes a caspase‐independent and RIPK3‐mediated cell death in both myoblasts and myotubes. In vivo, RIPK3‐deficient animals present improved body and muscle weights, a reduction of the aberrant activation of the DUX4 network genes, and an improvement of muscle histology. Conclusions These results provide evidence for a role of RIPK3 in DUX4‐mediated cell death and open new avenues of research.

Amyotrophic lateral sclerosis is a disease affecting upper and lower motor neurons. Although motor neuron death is the core event of ALS pathology, it is increasingly recognized that other tissues and cell types are affected in the disease, making potentially major contributions to the occurrence and progression of pathology. We review here the known cellular and molecular characteristics of muscle tissue affected by ALS. Evidence of toxicity in skeletal muscle tissue is considered, including metabolic dysfunctions, impaired proteostasis, and deficits in muscle regeneration and RNA metabolism. The role of muscle as a secretory organ, and effects on the skeletal muscle secretome are also covered, including the increase in secretion of toxic factors or decrease in essential factors that have consequences for neuronal function and survival.

Amyotrophic Lateral Sclerosis (ALS), Spinal Bulbar Muscular Atrophy (SBMA), and Spinal Muscular Atrophy (SMA) are motor neuron diseases (MNDs) characterised by progressive motor neuron degeneration, weakness and muscular atrophy. Lipid dysregulation is well recognised in each of these conditions and occurs prior to neurodegeneration. Several lipid markers have been shown to predict prognosis in ALS. Sphingolipids are complex lipids enriched in the central nervous system and are integral to key cellular functions including membrane stability and signalling pathways, as well as being mediators of neuroinflammation and neurodegeneration. This review highlights the metabolism of sphingomyelin (SM), the most abundant sphingolipid, and of its metabolite ceramide, and its role in the pathophysiology of neurodegeneration, focusing on MNDs. We also review published lipidomic studies in MNDs. In the 13 studies of patients with ALS, 12 demonstrated upregulation of multiple SM species and 6 demonstrated upregulation of ceramides. SM species also correlated with markers of clinical progression in five of six studies. These data highlight the potential use of SM and ceramide as biomarkers in ALS. Finally, we review potential therapeutic strategies for targeting sphingolipid metabolism in neurodegeneration.

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., Graphical Abstract Graphical AbstracteSkip-Finder uses information on exon skipping antisense oligonucleotides from the literature to produce a database and a skipping efficacy predictive tool to aid researchers in designing effective exon skipping therapies.

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder caused by mutations in the DMD gene and the subsequent lack of dystrophin protein. Recently, phosphorodiamidate morpholino oligomer (PMO)-antisense oligonucleotides (ASOs) targeting exon 51 or 53 to reestablish the DMD reading frame have received regulatory approval as commercially available drugs. However, their applicability and efficacy remain limited to particular patients. Large animal models and exon skipping evaluation are essential to facilitate ASO development together with a deeper understanding of dystrophinopathies. Using recombinant adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer, we generated a Yucatan miniature pig model of DMD with an exon 52 deletion mutation equivalent to one of the most common mutations seen in patients. Exon 52-deleted mRNA expression and dystrophin deficiency were confirmed in the skeletal and cardiac muscles of DMD pigs. Accordingly, dystrophin-associated proteins failed to be recruited to the sarcolemma. The DMD pigs manifested early disease onset with severe bodywide skeletal muscle degeneration and with poor growth accompanied by a physical abnormality, but with no obvious cardiac phenotype. We also demonstrated that in primary DMD pig skeletal muscle cells, the genetically engineered exon-52 deleted pig DMD gene enables the evaluation of exon 51 or 53 skipping with PMO and its advanced technology, peptide-conjugated PMO. The results show that the DMD pigs developed here can be an appropriate large animal model for evaluating in vivo exon skipping efficacy.

The rapid increase in the number of genetic variants identified to be associated with Amyotrophic Lateral Sclerosis (ALS) through genome-wide association studies (GWAS) has created an emerging need to understand the functional pathways that are implicated in the pathology of ALS. Gene-set analysis (GSA) is a powerful method that can provide insight into the associated biological pathways, determining the joint effect of multiple genetic markers. The main contribution of this review is the collection of ALS GSA studies that employ GWAS or individual-based genotype data, investigating their methodology and results related to ALS-associated molecular pathways. Furthermore, the limitations in standard single-gene analyses are summarized, highlighting the power of gene-set analysis, and a brief overview of the statistical properties of gene-set analysis and related concepts is provided. The main aims of this review are to investigate the reproducibility of the collected studies and identify their strengths and limitations, in order to enhance the experimental design and therefore the quality of the results of future studies, deepening our understanding of this devastating disease.

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.

Quality control of genomic data is an essential but complicated multi-step procedure, often requiring separate installation and expert familiarity with a combination of different bioinformatics tools. Software incompatibilities, and inconsistencies across computing environments, are recurrent challenges, leading to poor reproducibility. Existing semi-automated or automated solutions lack comprehensive quality checks, flexible workflow architecture, and user control. To address these challenges, we have developed snpQT: a scalable, stand-alone software pipeline using nextflow and BioContainers, for comprehensive, reproducible and interactive quality control of human genomic data. snpQT offers some 36 discrete quality filters or correction steps in a complete standardised pipeline, producing graphical reports to demonstrate the state of data before and after each quality control procedure. This includes human genome build conversion, population stratification against data from the 1,000 Genomes Project, automated population outlier removal, and built-in imputation with its own pre- and post- quality controls. Common input formats are used, and a synthetic dataset and comprehensive online tutorial are provided for testing, educational purposes, and demonstration. The snpQT pipeline is designed to run with minimal user input and coding experience; quality control steps are implemented with numerous user-modifiable thresholds, and workflows can be flexibly combined in custom combinations. snpQT is open source and freely available at https://github.com/nebfield/snpQT. A comprehensive online tutorial and installation guide is provided through to GWAS (https://snpqt.readthedocs.io/en/latest/), introducing snpQT using a synthetic demonstration dataset and a real-world Amyotrophic Lateral Sclerosis SNP-array dataset.

The field of neuromuscular research has seen considerable recent advances in the molecular and cellular understanding of muscle biology, and the treatment of neuromuscular disease [...]

BackgroundThe cause of the motor neuron (MN) death that drives terminal pathology in Amyotrophic Lateral Sclerosis (ALS) remains unknown, and it is thought that the cellular environment of the MN may play a key role in MN survival. Several lines of evidence implicate vesicles in ALS, including that extracellular vesicles may carry toxic elements from astrocytes towards motor neurons, and that pathological proteins have been identified in circulating extracellular vesicles of sporadic ALS patients. Since MN degeneration at the neuromuscular junction is a feature of ALS, and muscle is a vesicle-secretory tissue, we hypothesized that muscle vesicles may be involved in ALS pathology.MethodsSporadic ALS patients were confirmed to be ALS according to El Escorial criteria, were genotyped to test for classic gene mutations associated with ALS, and physical function was assessed using the ALSFRS-R score. Muscle biopsies of either mildly affected deltoids of ALS patients (n=27) or deltoids of aged-matched healthy subjects (n=30) were used for extraction of muscle stem cells, to perform immunohistology, or for electron microscopy. Muscle stem cells were characterized by immunostaining, RTqPCR and transcriptomic analysis. Secreted muscle vesicles were characterized by proteomic analysis, Western blot, NanoSight, and electron microscopy. The effects of muscle vesicles isolated from the culture medium of ALS and healthy myotubes were tested on healthy human-derived iPSC motor neurons and on healthy human myotubes, with untreated cells used as controls.ResultsAn accumulation of multivesicular bodies was observed in muscle biopsies of sporadic ALS patients by immunostaining and electron microscopy. Study of muscle biopsies and biopsy-derived denervation-naïve differentiated muscle stem cells (myotubes) revealed a consistent disease signature in ALS myotubes, including intracellular accumulation of exosome-like vesicles and disruption of RNA-processing. Compared to vesicles from healthy control myotubes, when administered to healthy motor neurons the vesicles of ALS myotubes induced shortened, less branched neurites, cell death, and disrupted localization of RNA and RNA-processing proteins. The RNA-processing protein FUS and a majority of its binding partners were present in ALS muscle vesicles, and toxicity was dependent on the expression level of FUS in recipient cells. Toxicity to recipient motor neurons was abolished by anti-CD63 immuno-blocking of vesicle uptake.ConclusionALS muscle vesicles are shown to be toxic to motor neurons, which establishes the skeletal muscle as a potential source of vesicle-mediated toxicity in ALS.One Sentence SummaryMuscle cells of ALS patients secrete vesicles that are toxic to motor neurons

Amyotrophic Lateral Sclerosis (ALS) is the most common late-onset motor neuron disorder, but our current knowledge of the molecular mechanisms and pathways underlying this disease remain elusive. This review (1) systematically identifies machine learning studies aimed at the understanding of the genetic architecture of ALS, (2) outlines the main challenges faced and compares the different approaches that have been used to confront them, and (3) compares the experimental designs and results produced by those approaches and describes their reproducibility in terms of biological results and the performances of the machine learning models. The majority of the collected studies incorporated prior knowledge of ALS into their feature selection approaches, and trained their machine learning models using genomic data combined with other types of mined knowledge including functional associations, protein-protein interactions, disease/tissue-specific information, epigenetic data, and known ALS phenotype-genotype associations. The importance of incorporating gene-gene interactions and cis-regulatory elements into the experimental design of future ALS machine learning studies is highlighted. Lastly, it is suggested that future advances in the genomic and machine learning fields will bring about a better understanding of ALS genetic architecture, and enable improved personalized approaches to this and other devastating and complex diseases.

Amyotrophic lateral sclerosis (ALS) is a terminal late-onset condition characterized by the loss of upper and lower motor neurons. Mutations in more than 30 genes are associated to the disease, but these explain only ~20% of cases. The molecular functions of these genes implicate a wide range of cellular processes in ALS pathology, a cohesive understanding of which may provide clues to common molecular mechanisms across both familial (inherited) and sporadic cases and could be key to the development of effective therapeutic approaches. Here, the different pathways that have been investigated in ALS are summarized, discussing in detail: mitochondrial dysfunction, oxidative stress, axonal transport dysregulation, glutamate excitotoxicity, endosomal and vesicular transport impairment, impaired protein homeostasis, and aberrant RNA metabolism. This review considers the mechanistic roles of ALS-associated genes in pathology, viewed through the prism of shared molecular pathways.

Background High-throughput transcriptomics measures mRNA levels for thousands of genes in a biological sample. Most gene expression studies aim to identify genes that are differentially expressed between different biological conditions, such as between healthy and diseased states. However, these data can also be used to identify genes that are co-expressed within a biological condition. Gene co-expression is used in a guilt-by-association approach to prioritize candidate genes that could be involved in disease, and to gain insights into the functions of genes, protein relations, and signaling pathways. Most existing gene co-expression databases are generic, amalgamating data for a given organism regardless of tissue-type. Methods To study muscle-specific gene co-expression in both normal and pathological states, publicly available gene expression data were acquired for 2376 mouse and 2228 human striated muscle samples, and separated into 142 categories based on species (human or mouse), tissue origin, age, gender, anatomic part, and experimental condition. Co-expression values were calculated for each category to create the MyoMiner database. Results Within each category, users can select a gene of interest, and the MyoMiner web interface will return all correlated genes. For each co-expressed gene pair, adjusted p-value and confidence intervals are provided as measures of expression correlation strength. A standardized expression-level scatterplot is available for every gene pair r-value. MyoMiner has two extra functions: (a) a network interface for creating a 2-shell correlation network, based either on the most highly correlated genes or from a list of genes provided by the user with the option to include linked genes from the database and (b) a comparison tool from which the users can test whether any two correlation coefficients from different conditions are significantly different. Conclusions These co-expression analyses will help investigators to delineate the tissue-, cell-, and pathology-specific elements of muscle protein interactions, cell signaling and gene regulation. Changes in co-expression between pathologic and healthy tissue may suggest new disease mechanisms and help define novel therapeutic targets. Thus, MyoMiner is a powerful muscle-specific database for the discovery of genes that are associated with related functions based on their co-expression. MyoMiner is freely available at https://www.sys-myo.com/myominer

Dysferlinopathy is a progressive myopathy caused by mutations in the dysferlin (DYSF) gene. Dysferlin protein plays a major role in plasma-membrane resealing. Some patients with DYSF deletion mutations exhibit mild symptoms, suggesting some regions of DYSF can be removed without significantly impacting protein function. Antisense-mediated exon-skipping therapy uses synthetic molecules called antisense oligonucleotides to modulate splicing, allowing exons harboring or near genetic mutations to be removed and the open reading frame corrected. Previous studies have focused on DYSF exon 32 skipping as a potential therapeutic approach, based on the association of a mild phenotype with the in-frame deletion of exon 32. To date, no other DYSF exon-skipping targets have been identified, and the relationship between DYSF exon deletion pattern and protein function remains largely uncharacterized. In this study, we utilized a membrane-wounding assay to evaluate the ability of plasmid constructs carrying mutant DYSF, as well as antisense oligonucleotides, to rescue membrane resealing in patient cells. We report that multi-exon skipping of DYSF exons 26–27 and 28–29 rescues plasma-membrane resealing. Successful translation of these findings into the development of clinical antisense drugs would establish new therapeutic approaches that would be applicable to ∼5%–7% (exons 26–27 skipping) and ∼8% (exons 28–29 skipping) of dysferlinopathy patients worldwide. Keywords: exon skipping, antisense, morpholino, dysferlin, dysferlinopathy, limb-girdle muscular dystrophy type 2B, Miyoshi myopathy, distal myopathy, plasma membrane, membrane wounding

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder caused by mutations in the DMD gene and the subsequent lack of dystrophin protein. Recently, phosphorodiamidate morpholino oligomer (PMO)-antisense oligonucleotides (ASOs) targeting exon 51 or 53 to reestablish the DMD reading frame have received regulatory approval as commercially available drugs. However, their applicability and efficacy remain limited to particular patients. Large animal models and exon skipping evaluation are essential to facilitate ASO development together with a deeper understanding of dystrophinopathies. Using recombinant adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer, we generated a Yucatan miniature pig model of DMD with an exon 52 deletion mutation equivalent to one of the most common mutations seen in patients. Exon 52-deleted mRNA expression and dystrophin deficiency were confirmed in the skeletal and cardiac muscles of DMD pigs. Accordingly, dystrophin-associated proteins failed to be recruited to the sarcolemma. The DMD pigs manifested early disease onset with severe bodywide skeletal muscle degeneration and with poor growth accompanied by a physical abnormality, but with no obvious cardiac phenotype. We also demonstrated that in primary DMD pig skeletal muscle cells, the genetically engineered exon-52 deleted pig DMD gene enables the evaluation of exon 51 or 53 skipping with PMO and its advanced technology, peptide-conjugated PMO. The results show that the DMD pigs developed here can be an appropriate large animal model for evaluating in vivo exon skipping efficacy.

Intercellular communication between neurons and their surrounding cells occurs through the secretion of soluble molecules or release of vesicles such as exosomes into the extracellular space, participating in brain homeostasis. Under neuro-degenerative conditions associated with ageing, such as amyotrophic lateral sclerosis (ALS), Alzheimer’s or Parkinson’s disease, exosomes are suspected to propagate toxic proteins. The topic of this review is the role of exosomes in ageing conditions and more specifically in ALS. Our current understanding of exosomes and exosome-related mechanisms is first summarized in a general sense, including their biogenesis and secretion, heterogeneity, cellular interaction and intracellular fate. Their role in the Central Nervous System (CNS) and ageing of the neuromotor system is then considered in the context of exosome-induced signaling. The review then focuses on exosomes in age-associated neurodegenerative disease. The role of exosomes in ALS is highlighted, and their use as potential biomarkers to diagnose and prognose ALS is presented. The therapeutic implications of exosomes for ALS are considered, whether as delivery vehicles, neurotoxic targets or as corrective drugs in and of themselves. A diverse set of mechanisms underpin the functional roles, both confirmed and potential, of exosomes, generally in ageing and specifically in motor neurone disease. Aspects of their contents, biogenesis, uptake and modifications offer many plausible routes towards the development of novel biomarkers and therapeutics.

Summary Gene coexpression analysis refers to the discovery of sets of genes which exhibit similar expression patterns across multiple transcriptomic data sets, such as microarray experiment data of public repositories. Arabidopsis Coexpression Tool (ACT), a gene coexpression analysis web tool for Arabidopsis thaliana, identifies genes which are correlated to a driver gene. Primary microarray data from ATH1 Affymetrix platform were processed with Single-Channel Array Normalization algorithm and combined to produce a coexpression tree which contains ∼21,000 A. thaliana genes. ACT was developed to present subclades of coexpressed genes, as well as to perform gene set enrichment analysis, being unique in revealing enriched transcription factors targeting coexpressed genes. ACT offers a simple and user-friendly interface producing working hypotheses which can be experimentally verified for the discovery of gene partnership, pathway membership, and transcriptional regulation. ACT analyses have been successful in identifying not only genes with coordinated ubiquitous expressions but also genes with tissue-specific expressions., Graphical abstract, Highlights • 3500 primary microarray data from ATH1 Affymetrix chip were normalized using SCAN • A tree with 21,287 Arabidopsis thaliana gene coexpression was constructed using UPGMA • ACT is a web tool that identifies genes which are coexpressed to a driver gene • ACT performs biological term enrichment analysis in coexpressed gene tree clades, Plant genetics; Bioinformatics; Plant bioinformatics

International audience; 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.

Repair of skeletal muscle after sarcolemmal damage involves dysferlin and dysferlin-interacting proteins such as annexins. Mice and patient lacking dysferlin exhibit chronic muscle inflammation and adipogenic replacement of the myofibers. Here, we show that similar to dysferlin, lack of annexin A2 (AnxA2) also results in poor myofiber repair and progressive muscle weakening with age. By longitudinal analysis of AnxA2-deficient muscle we find that poor myofiber repair due to the lack of AnxA2 does not result in chronic inflammation or adipogenic replacement of the myofibers. Further, deletion of AnxA2 in dysferlin deficient mice reduced muscle inflammation, adipogenic replacement of myofibers, and improved muscle function. These results identify multiple roles of AnxA2 in muscle repair, which includes facilitating myofiber repair, chronic muscle inflammation and adipogenic replacement of dysferlinopathic muscle. It also identifies inhibition of AnxA2-mediated inflammation as a novel therapeutic avenue for treating muscle loss in dysferlinopathy.

Amyotrophic lateral sclerosis (ALS), also known as motor neurone disease, is an incurable neurodegenerative condition, characterized by the loss of upper and lower motor neurons. It affects 1-1.8/100,000 individuals worldwide, and the number of cases is projected to increase as the population ages. Thus, there is an urgent need to identify both therapeutic targets and disease-specific biomarkers - biomarkers that would be useful to diagnose and stratify patients into different sub-groups for therapeutic strategies, as well as biomarkers to follow the efficacy of any treatment tested during clinical trials. There is a lack of knowledge about pathogenesis and many hypotheses. Numerous ‘omics’ studies have been conducted on ALS in the past decade to identify a disease-signature in tissues and circulating biomarkers. The first goal of the present review was to group the molecular pathways that have been implicated in monogenic forms of ALS, to enable the description of patient strata corresponding to each pathway grouping. This strategy allowed us to suggest 14 strata, each potentially targetable by different pharmacological strategies. The second goal of this review was to identify diagnostic/prognostic biomarker candidates consistently observed across the literature. For this purpose, we explore previous biomarker-relevant ‘omics’ studies of ALS and summarize their findings, focusing on potential circulating biomarker candidates. We systematically review 118 papers on biomarkers published during the last decade. Several candidate markers were consistently shared across the results of different studies in either cerebrospinal fluid (CSF) or blood (leukocyte or serum/plasma). Although these candidates still need to be validated in a systematic manner, we suggest the use of combinations of biomarkers that would likely reflect the “health status” of different tissues, including motor neuron health (e.g. pNFH and NF-L, cystatin C, Transthyretin), inflammation status (e.g. MCP-1, miR451), muscle health (miR-338-3p, miR-206) and metabolism (homocysteine, glutamate, cholesterol). In light of these studies and because ALS is increasingly perceived as a multi-system disease, the identification of a panel of biomarkers that accurately reflect features of pathology is a priority, not only for diagnostic purposes but also for prognostic or predictive applications.

Background The approach of building large collections of gene sets and then systematically testing hypotheses across these collections is a powerful tool in functional genomics, both in the pathway analysis of omics data and to uncover the polygenic effects associated with complex diseases in genome-wide association study. The Molecular Signatures Database includes collections of oncogenic and immunologic signatures enabling researchers to compare transcriptional datasets across hundreds of previous studies and leading to important insights in these fields, but such a resource does not currently exist for neuromuscular research. In previous work, we have shown the utility of gene set approaches to understand muscle cell physiology and pathology. Methods Following a systematic survey of public muscle data, we passed gene expression profiles from 4305 samples through a robust pre-processing and standardized data analysis pipeline. Two hundred eighty-two samples were discarded based on a battery of rigorous global quality controls. From among the remaining studies, 578 comparisons of interest were identified by a combination of text mining and manual curation of the study meta-data. For each comparison, significantly dysregulated genes (FDR adjusted p

Multiple genes and mechanisms of pathophysiology have been implicated in amyotrophic lateral sclerosis (ALS), suggesting it is a complex systemic disease. With this in mind, applying personalized medicine (PM) approaches to tailor treatment pipelines for ALS patients may be necessary. The modelling and analysis of molecular interaction networks could represent valuable resources in defining ALS-associated pathways and discovering novel therapeutic targets. Here we review existing omics datasets and analytical approaches, in order to consider how molecular interaction networks could improve our understanding of the molecular pathophysiology of this fatal neuromuscular disorder.

Antisense oligonucleotide-mediated exon skipping is a promising therapeutic approach for the treatment of various genetic diseases and a therapy which has gained significant traction in recent years following FDA approval of new antisense-based drugs. Exon skipping for Duchenne muscular dystrophy (DMD) works by modulating dystrophin pre-mRNA splicing, preventing incorporation of frame-disrupting exons into the final mRNA product while maintaining the open reading frame, to produce a shortened-yet-functional protein as seen in milder Becker muscular dystrophy (BMD) patients. Exons 45-55 skipping in dystrophin is potentially applicable to approximately 47% of DMD patients because many mutations occur within this "mutation hotspot." In addition, patients naturally harboring a dystrophin exons 45-55 in-frame deletion mutation have an asymptomatic or exceptionally mild phenotype compared to shorter in-frame deletion mutations in this region. As such, exons 45-55 skipping could transform the DMD phenotype into an asymptomatic or very mild BMD phenotype and rescue nearly a half of DMD patients. In addition, this strategy is potentially applicable to some BMD patients as well, who have in-frame deletion mutations in this region. As the degree of exon skipping correlates with therapeutic outcomes, reliable measurements of exon skipping efficiencies are essential to the development of novel antisense-mediated exon skipping therapeutics. In the case of DMD, researchers have often relied upon human muscle fibers obtained from muscle biopsies for testing; however, this method is highly invasive and patient myofibers can display limited proliferative ability. To overcome these challenges, researchers can generate myofibers from patient fibroblast cells by transducing the cells with a viral vector containing MyoD, a myogenic regulatory factor. Here, we describe a methodology for assessing dystrophin exons 45-55 multiple skipping efficiency using antisense oligonucleotides in human muscle cells derived from DMD patient fibroblast cells.

Duchenne muscular dystrophy results from loss of the protein dystrophin, which links the intracellular cytoskeletal network with the extracellular matrix, but deficiency in this function does not fully explain the onset or progression of the disease. While some intracellular events involved in the degeneration of dystrophin-deficient muscle fibers have been well characterized, changes in their secretory profile are undescribed. To analyze the secretome profile of mdx myotubes independently of myonecrosis, we labeled the proteins of mdx and wild-type myotubes with stable isotope-labeled amino acids (SILAC), finding marked enrichment of vesicular markers in the mdx secretome. These included the lysosomal-associated membrane protein, LAMP1, that co-localized in vesicles with an over-secreted cytoskeletal protein, myosin light chain 1. These LAMP1/MLC1-3-positive vesicles accumulated in the cytosol of mdx myotubes and were secreted into the culture medium in a range of abnormal densities. Restitution of dystrophin expression, by exon skipping, to some 30 % of the control value, partially normalized the secretome profile and the excess LAMP1 accumulation. Together, our results suggest that a lack of dystrophin leads to a general dysregulation of vesicle trafficking. We hypothesize that disturbance of the export of proteins through vesicles occurs before, and then concurrently with, the myonecrotic cascade and contributes chronically to the pathophysiology of DMD, thereby presenting us with a range of new potential therapeutic targets.

Background hTERT/cdk4 immortalized myogenic human cell lines represent an important tool for skeletal muscle research, being used as therapeutically pertinent models of various neuromuscular disorders and in numerous fundamental studies of muscle cell function. However, the cell cycle is linked to other cellular processes such as integrin regulation, the PI3K/Akt pathway, and microtubule stability, raising the question as to whether genetic modification related to the cell cycle results in secondary effects that could undermine the validity of these cell models. Results Here we subjected five healthy and disease muscle cell isolates to transcriptomic analysis, comparing immortalized lines with their parent primary populations in both differentiated and undifferentiated states, and testing their myogenic character by comparison with non-myogenic (CD56-negative) cells. Principal component analysis of global gene expression showed tight clustering of immortalized myoblasts to their parent primary populations, with clean separation from the non-myogenic reference. Comparison was made to publicly available transcriptomic data from studies of muscle human pathology, cell, and animal models, including to derive a consensus set of genes previously shown to have altered regulation during myoblast differentiation. Hierarchical clustering of samples based on gene expression of this consensus set showed that immortalized lines retained the myogenic expression patterns of their parent primary populations. Of 2784 canonical pathways and gene ontology terms tested by gene set enrichment analysis, none were significantly enriched in immortalized compared to primary cell populations. We observed, at the whole transcriptome level, a strong signature of cell cycle shutdown associated with senescence in one primary myoblast population, whereas its immortalized clone was protected. Conclusions Immortalization had no observed effect on the myogenic cascade or on any other cellular processes, and it was protective against the systems level effects of senescence that are observed at higher division counts of primary cells. Electronic supplementary material The online version of this article (doi:10.1186/s13395-016-0115-5) contains supplementary material, which is available to authorized users.

Duchenne muscular dystrophy (DMD), one of the most common and lethal genetic disorders, is caused by mutations of the dystrophin gene. Removal of an exon or of multiple exons using antisense molecules has been demonstrated to allow synthesis of truncated 'Becker muscular dystrophy-like' dystrophin.Approximately 15% of DMD cases are caused by a nonsense mutation. Although patient databases have previously been surveyed for applicability to each deletion mutation pattern, this is not so for nonsense mutations. Here, we examine the world-wide database containing notations for more than 1200 patients with nonsense mutations. Approximately 47% of nonsense mutations can be potentially treated with single exon skipping, rising to 90% with double exon skipping, but to reach this proportion requires the development of exon skipping molecules targeting some 68 of dystrophin's 79 exons, with patient numbers spread thinly across those exons. In this review, we discuss progress and remaining hurdles in exon skipping and an alternative strategy, stop-codon readthrough.Antisense-mediated exon skipping therapy is targeted highly at the individual patient and is a clear example of personalized medicine. An efficient regulatory path for drug approval will be a key to success.

Antisense-mediated exon skipping has made significant progress as a therapeutic platform in recent years, especially in the case of Duchenne muscular dystrophy (DMD). Despite FDA approval of eteplirsen-the first-ever antisense drug clinically marketed for DMD-exon skipping therapy still faces the significant hurdles of limited applicability and unknown truncated protein function. In-frame exon skipping of dystrophin exons 45-55 represents a significant approach to treating DMD, as a large proportion of patients harbor mutations within this "hotspot" region. Additionally, patients harboring dystrophin exons 45-55 deletion mutations are reported to have exceptionally mild to asymptomatic phenotypes. Here, we demonstrate that a cocktail of phosphorodiamidate morpholino oligomers can effectively skip dystrophin exons 45-55 in vitro in myotubes transdifferentiated from DMD patient fibroblast cells. This is the first report of substantive exons 45-55 skipping in DMD patient cells. These findings help validate the use of transdifferentiated patient fibroblast cells as a suitable cell model for dystrophin exon skipping assays and further emphasize the feasibility of dystrophin exons 45-55 skipping in patients.

Many of the mutations associated with Duchenne muscular dystrophy can potentially be rescued by exon-skipping therapy, targeting selected exons of prespliced mRNA for the dystrophin gene with antisense oligonucleotides, thereby restoring reading frames. The recent development of antisense oligonucleotides with higher stability and lower toxicity, such as morpholinos, has made it possible to restore dystrophin efficiently in dystrophic mice in vivo with no obvious side effects. There seems little doubt that such exon-skipping therapy is destined to proceed to the clinical application stage in patients with Duchenne muscular dystrophy. One of the remaining issues to be addressed is the skipping of multiple exons because such multi-exon skipping therapy could expand the potential patient target population to include 80% of those with duplication mutations and 90% of those with deletion mutations. At present, this multi-exon skipping strategy is being investigated in dystrophic dogs as well as dystrophic mice. There are several challenges that still need to be overcome, including the low uptake of antisense oligonucleotides into the heart and the need to design efficient, nontoxic, cost-effective oligonucleotides. This review summarizes recent progress in exon-skipping therapy and discusses future perspectives with regard to human clinical trials.

International audience; The molecular mechanisms by which aging affects stem cell number and function are poorly understood. Murine data have implicated cellular senescence in the loss of muscle stem cells with aging. Here, using human cells and by carrying out experiments within a strictly pre-senescent division count, we demonstrate an impaired capacity for stem cell self-renewal in elderly muscle. We link aging to an increased methylation of the SPRY1 gene, a known regulator of muscle stem cell quiescence. Replenishment of the reserve cell pool was modulated experimentally by demethylation or siRNA knockdown of SPRY1. We propose that suppression of SPRY1 by age-associated methylation in humans inhibits the replenishment of the muscle stem cell pool, contributing to a decreased regenerative response in old age. We further show that aging does not affect muscle stem cell senescence in humans.

International audience; The use of antisense ‘splice-switching’ oligonucleotides to induce exon skipping represents a potential therapeutic approach to various human genetic diseases. It has achieved greatest maturity in exon skipping of the dystrophin transcript in Duchenne muscular dystrophy (DMD), for which several clinical trials are completed or ongoing, and a large body of data exists describing tested oligonucleotides and their efficacy. The rational design of an exon skipping oligonucleotide involves the choice of an antisense sequence, usually between 15 and 32 nucleotides, targeting the exon that is to be skipped. Although parameters describing the target site can be computationally estimated and several have been identified to correlate with efficacy, methods to predict efficacy are limited. Here, an in silico pre-screening approach is proposed, based on predictive statistical modelling. Previous DMD data were compiled together and, for each oligonucleotide, some 60 descriptors were considered. Statistical modelling approaches were applied to derive algorithms that predict exon skipping for a given target site. We confirmed (1) the binding energetics of the oligonucleotide to the RNA, and (2) the distance in bases of the target site from the splice acceptor site, as the two most predictive parameters, and we included these and several other parameters (while discounting many) into an in silico screening process, based on their capacity to predict high or low efficacy in either phosphorodiamidate morpholino oligomers (89% correctly predicted) and/or 2’O Methyl RNA oligonucleotides (76% correctly predicted). Predictions correlated strongly with in vitro testing for sixteen de novo PMO sequences targeting various positions on DMD exons 44 (R2 0.89) and 53 (R2 0.89), one of which represents a potential novel candidate for clinical trials. We provide these algorithms together with a computational tool that facilitates screening to predict exon skipping efficacy at each position of a target exon.

Background Preclinical testing of potential therapies for Duchenne muscular dystrophy (DMD) is conducted predominantly of the mdx mouse. But lack of a detailed quantitative description of the pathology of this animal limits our ability to evaluate the effectiveness of putative therapies or their relevance to DMD. Methods Accordingly, we have measured the main cellular components of muscle growth and regeneration over the period of postnatal growth and early pathology in mdx and wild-type (WT) mice; phalloidin binding is used as a measure of fibre size, myonuclear counts and BrdU labelling as records of myogenic activity. Results We confirm a two-phase postnatal growth pattern in WT muscle: first, increase in myonuclear number over weeks 1 to 3, then expansion of myonuclear domain. Mdx muscle growth lags behind that of WT prior to overt signs of pathology. Fibres are smaller, with fewer myonuclei and smaller myonuclear domains. Moreover, satellite cells are more readily detached from mdx than WT muscle fibres. At 3 weeks, mdx muscles enter a phase of florid myonecrosis, accompanied by concurrent regeneration of an intensity that results in complete replacement of pre-existing muscle over the succeeding 3 to 4 weeks. Both WT and mdx muscles attain maximum size by 12 to 14 weeks, mdx muscle fibres being up to 50% larger than those of WT as they become increasingly branched. Mdx muscle fibres also become hypernucleated, containing twice as many myonuclei per sarcoplasmic volume, as those of WT, the excess corresponding to the number of centrally placed myonuclei. Conclusions The best-known consequence of lack of dystrophin that is common to DMD and the mdx mouse is the conspicuous necrosis and regeneration of muscle fibres. We present protocols for measuring this in terms both of loss of muscle nuclei previously labelled with BrdU and of the intensity of myonuclear labelling with BrdU administered during the regeneration period. Both measurements can be used to assess the efficacy of putative antinecrotic agents. We also show that lack of dystrophin is associated with a number of previously unsuspected abnormalities of muscle fibre structure and function that do not appear to be directly associated with myonecrosis.