Muscle Gene Sets: a versatile methodological aid to functional genomics in the neuromuscular field

International audienceBACKGROUND: 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 < 0.05) were identified.RESULTS: Lists of dysregulated genes were divided between upregulated and downregulated to give 1156 Muscle Gene Sets (MGS). This resource is available for download ( www.sys-myo.com/muscle_gene_sets ) and is accessible through three commonly used functional genomics platforms (GSEA, EnrichR, and WebGestalt). Basic guidance and recommendations are provided for the use of MGS through these platforms. In addition, consensus muscle gene sets were created to capture the overlap between the results of similar studies, and analysis of these highlighted the potential for novel disease-relevant findings.CONCLUSIONS: The MGS resource can be used to investigate the behaviour of any list of genes across previous comparisons of muscle conditions, to compare previous studies to one another, and to explore the functional relationship of muscle dysregulation to the Gene Ontology. Its major intended use is in enrichment testing for functional genomics analysis

Genome-Wide Gene-Set Analysis Approaches in Amyotrophic Lateral Sclerosis

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

The Cellular and Molecular Signature of ALS in Muscle

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

The geometry of hydrogen-bonds and carbonyl-carbonyl interactions between trans-amides in proteins and small molecules

The geometries, at the level of atoms or groups of atoms, of interactions and motifs found in crystal structures in the Protein Data Bank (PDB) and the Cambridge Structural Database (CSD) are analysed. The bulk of the thesis examines electrostatic interactions that occur between trans secondary amide groups, or peptide groups as they are known in proteins. The two types considered are N-H-"O=C hydrogen-bonds and carbonyl-carbonyl interactions. Additionally, a chapter investigates specific hydrogen-bonded motifs, known as asx- and ST-tums, that commonly occur within protein structures. The geometry of hydrogen-bonds between trans secondary amide groups (i.e. peptide groups) has been studied extensively in proteins, and to a lesser extent in the CSD. They are a subset of the general case of N-H-O=C hydrogen-bonds. Previous analyses of the CSD have shown a tendency for N-H"-O=C hydrogen-bonds to exhibit lone-pair directionality, where the hydrogen atom is near to the plane of the lone-pairs of the carbonyl oxygen atom, and the H-O=C angle approaches 120°. For trans secondary amides the in-plane preference is also observed, but the H-''O=C angle is greater, averaging about 150°. Here, an examination of the CSD allows elucidation of four factors that together account for this difference in H-"O=C: 1. A smaller proportion of trans secondary amide carbonyl oxygens accept more than one hydrogen-bond than do carbonyl oxygens in general. 2. N-H"-O=C bonds often occur in 'ring' motifs with relatively constrained geometries and H"-O=C values near 120°. These cannot be formed by trans secondary amides. 3. Chains of hydrogen-bonds between trans secondary amides, with large H-O=C values, often extend throughout the crystal lattice. 4. The steric accessibility of trans secondary amide carbonyl oxygens is less than for carbonyl oxygens in general. It has been suggested that electrostatic interactions between carbonyl groups affect hydrogen-bond geometry in alpha-helix and beta-sheet, influence the twist of P-sheet, encourage polarization of carbonyl groups in alpha-helix, and explain the propensity of asparagine and aspartate residues in unusual regions of the Ramachandran plot. The carbonyl groups of ketones in the CSD frequently interact with each other, and commonly occur in three geometric motifs, that can be described as antiparallel, parallel, or perpendicular. At optimal geometry the antiparallel motif has energetic favourability approaching that of a medium-strength hydrogen-bond. Here it is shown that, after hydrogen-bonding has been taken into consideration, carbonyl-carbonyl interactions between trans secondary amides in the CSD also occur in these three motifs, and with a surprisingly similar propensity: 48% occur in the antiparallel arrangement (cf. 49% of those between ketones). Furthermore, interactions between main-chain carbonyl groups in a 454-chain subset of the PDB are identified. For each carbonyl-carbonyl interaction, its geometry, local secondary structure, and local hydrogen-bonding, are considered. The three motifs present in the CSD are not found to be representative of the geometries present in the PDB. However, other favourable carbonyl-carbonyl interaction motifs are observed. These occur in a variety of situations with respect to secondary structure and hydrogen-bonding, and are prevalent at the C-termini of alpha-helices. They are shown to contribute to the stability of common C-termini capping conformations, one being the Schellman loop, the other being the case where a proline terminates the helix. The hydrogen-bonded beta-turn is a small, well characterised, protein motif defined by a hydrogen-bond between the main-chain carbonyl group of one residue and the main-chain N-H group of another three residues ahead in the polypeptide chain. There are four common types, distinguished by geometry: I, I', II, and II'. In Asx- and ST-turns, the side-chain carbonyl of an asparagine, aspartate, serine, or threonine residue hydrogen-bonds with the main-chain N-H of a residue two ahead in the chain, such that they structurally mimic the beta-turn. Asx-turns have previously been categorized into four classes and ST-turns into three categories, based on side-chain rotamer types and the conformation of the central residue of each turn. Here it is shown that the four classes of asx-turn are geometrically equivalent to the four types of hydrogen-bonded beta-turn, and that the three categories of ST-turn are geometrically equivalent to three of the four types of hydrogen-bonded beta-turn. It is proposed that asx- and ST-turns be named using the type I, II, I' and II' beta-turn nomenclature. Using this nomenclature, the frequency of occurrence of both asx- and ST-turns is: type IF > type I > type II > type F, whereas for p-turns it is type I > type II > type F > type IF. It is found that the type II Asx- or ST-turn is the same as a previously identified hydrogen-bonded motif called the Asx- or ST-nest

The isolated muscle fibre as a model of disuse atrophy: characterization using PhAct, a method to quantify f-actin

Research into muscle atrophy and hypertrophy is hampered by limitations of the available experimental models. Interpretation of in vivo experiments is confounded by the complexity of the environment while in vitro models are subject to the marked disparities between cultured myotubes and the mature myofibres of living tissues. Here we develop a method (PhAct) based on ex vivo maintenance of the isolated myofibre as a model of disuse atrophy, using standard microscopy equipment and widely available analysis software, to measure f-actin content per myofibre and per nucleus over two weeks of ex vivo maintenance. We characterize the 35% per week atrophy of the isolated myofibre in terms of early changes in gene expression and investigate the effects on loss of muscle mass of modulatory agents, including Myostatin and Follistatin. By tracing the incorporation of a nucleotide analogue we show that the observed atrophy is not associated with loss or replacement of myonuclei. Such a completely controlled investigation can be conducted with the myofibres of a single muscle. With this novel method we can identify those features and mechanisms of atrophy and hypertrophy that are intrinsic to the muscle fibre from those that include activities of other tissues and systemic agents

Identification of novel antisense-mediated exon skipping targets in DYSF for therapeutic treatment of dysferlinopathy

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 woundin

Exosomes in Ageing and Motor Neurone Disease: Biogenesis, Uptake Mechanisms, Modifications in Disease and Uses in the Development of Biomarkers and Therapeutics

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