Pilot investigations into the mechanistic basis for adverse effects of glucocorticoids in dysferlinopathy.

BACKGROUND: Dysferlinopathies are a clinically heterogeneous group of muscular dystrophies caused by gene mutations resulting in deficiency of the membrane-associated protein dysferlin. They manifest post-growth and are characterised by muscle wasting (primarily in the limb and limb-gridle muscles), inflammation, and replacement of myofibres with adipose tissue. The precise pathomechanism for dysferlinopathy is currently unclear; as such there are no treatments currently available. Glucocorticoids (GCs) are widely used to reduce inflammation and treat muscular dystrophies, but when administered to patients with dysferlinopathy, they have unexpected adverse effects, with accelerated loss of muscle strength. METHODS: To investigate the mechanistic basis for the adverse effects of GCs in dysferlinopathy, the potent GC dexamethasone (Dex) was administered for 4-5 weeks (0.5-0.75 µg/mL in drinking water) to dysferlin-deficient BLA/J and normal wild-type (WT) male mice, sampled at 5 (Study 1) or 10 months (Study 2) of age. A wide range of analyses were conducted. Metabolism- and immune-related gene expression was assessed in psoas muscles at both ages and in quadriceps at 10 months of age. For the 10-month-old mice, quadriceps and psoas muscle histology was assessed. Additionally, we investigated the impact of Dex on the predominantly slow and fast-twitch soleus and extensor digitorum longus (EDL) muscles (respectively) in terms of contractile function, myofibre-type composition, and levels of proteins related to contractile function and metabolism, plus glycogen. RESULTS: At both ages, many complement-related genes were highly expressed in BLA/J muscles, and WT mice were generally more responsive to Dex than BLA/J. The effects of Dex on BLA/J mice included (i) increased expression of inflammasome-related genes in muscles (at 5 months) and (ii) exacerbated histopathology of quadriceps and psoas muscles at 10 months. A novel observation was pronounced staining for glycogen in many myofibres of the damaged quadriceps muscles, with large pale vacuolated myofibres, suggesting possible myofibre death by oncosis. CONCLUSION: These pilot studies provide a new focus for further investigation into the adverse effects of GCs on dysferlinopathic muscles.

Oxidised Albumin Levels in Plasma and Skeletal Muscle as Biomarkers of Disease Progression and Treatment Efficacy in Dystrophic mdx Mice.

Redox modifications to the plasma protein albumin have the potential to be used as biomarkers of disease progression and treatment efficacy in pathologies associated with inflammation and oxidative stress. One such pathology is Duchenne muscular dystrophy (DMD), a fatal childhood disease characterised by severe muscle wasting. We have previously shown in the mdx mouse model of DMD that plasma albumin thiol oxidation is increased; therefore, the first aim of this paper was to establish that albumin thiol oxidation in plasma reflects levels within mdx muscle tissue. We therefore developed a method to measure tissue albumin thiol oxidation. We show that albumin thiol oxidation was increased in both mdx muscle and plasma, with levels correlated with measures of dystropathology. In dystrophic muscle, albumin content was associated with areas of myonecrosis. The second aim was to test the ability of plasma thiol oxidation to track acute changes in dystropathology: we therefore subjected mdx mice to a single treadmill exercise session (known to increase myonecrosis) and took serial blood samples. This acute exercise caused a transient increase in total plasma albumin oxidation and measures of dystropathology. Together, these data support the use of plasma albumin thiol oxidation as a biomarker to track active myonecrosis in DMD.

Considering the Promise of Vamorolone for Treating Duchenne Muscular Dystrophy.

This commentary provides an independent consideration of data related to the drug vamorolone (VBP15) as an alternative steroid proposed for treatment of Duchenne muscular dystrophy (DMD). Glucocorticoids such as prednisone and deflazacort have powerful anti-inflammatory benefits and are the standard of care for DMD, but their long-term use can result in severe adverse side effects; thus, vamorolone was designed as a unique dissociative steroidal anti-inflammatory drug, to retain efficacy and minimise these adverse effects. Extensive clinical trials (ongoing) have investigated the use of vamorolone for DMD, with two trials also for limb-girdle muscular dystrophies including dysferlinopathy (current), plus a variety of pre-clinical trials published. Vamorolone looks very promising, with similar efficacy and some reduced adverse effects (e.g., related to height) compared with other glucocorticoids, specifically prednisone/prednisolone, although it has not yet been directly compared with deflazacort. Of particular interest to clarify is the optimal clinical dose and other aspects of vamorolone that are proposed to provide additional benefits for membranes of dystrophic muscle: to stabilise and protect the sarcolemma from damage and enhance repair. The use of vamorolone (and other glucocorticoids) needs to be evaluated in terms of overall long-term efficacy and cost, and also in comparison with many candidate non-steroidal drugs with anti-inflammatory and other benefits for DMD.

Hallmarks of ageing in human skeletal muscle and implications for understanding the pathophysiology of sarcopenia in women and men.

Ageing is a complex biological process associated with increased morbidity and mortality. Nine classic, interdependent hallmarks of ageing have been proposed involving genetic and biochemical pathways that collectively influence ageing trajectories and susceptibility to pathology in humans. Ageing skeletal muscle undergoes profound morphological and physiological changes associated with loss of strength, mass, and function, a condition known as sarcopenia. The aetiology of sarcopenia is complex and whilst research in this area is growing rapidly, there is a relative paucity of human studies, particularly in older women. Here, we evaluate how the nine classic hallmarks of ageing: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication contribute to skeletal muscle ageing and the pathophysiology of sarcopenia. We also highlight five novel hallmarks of particular significance to skeletal muscle ageing: inflammation, neural dysfunction, extracellular matrix dysfunction, reduced vascular perfusion, and ionic dyshomeostasis, and discuss how the classic and novel hallmarks are interconnected. Their clinical relevance and translational potential are also considered.

Slow or fast: Implications of myofibre type and associated differences for manifestation of neuromuscular disorders.

Many neuromuscular disorders can have a differential impact on a specific myofibre type, forming the central premise of this review. The many different skeletal muscles in mammals contain a spectrum of slow- to fast-twitch myofibres with varying levels of protein isoforms that determine their distinctive contractile, metabolic, and other properties. The variations in functional properties across the range of classic 'slow' to 'fast' myofibres are outlined, combined with exemplars of the predominantly slow-twitch soleus and fast-twitch extensor digitorum longus muscles, species comparisons, and techniques used to study these properties. Other intrinsic and extrinsic differences are discussed in the context of slow and fast myofibres. These include inherent susceptibility to damage, myonecrosis, and regeneration, plus extrinsic nerves, extracellular matrix, and vasculature, examined in the context of growth, ageing, metabolic syndrome, and sexual dimorphism. These many differences emphasise the importance of carefully considering the influence of myofibre-type composition on manifestation of various neuromuscular disorders across the lifespan for both sexes. Equally, understanding the different responses of slow and fast myofibres due to intrinsic and extrinsic factors can provide deep insight into the precise molecular mechanisms that initiate and exacerbate various neuromuscular disorders. This focus on the influence of different myofibre types is of fundamental importance to enhance translation for clinical management and therapies for many skeletal muscle disorders.

Correction: Dysferlin-deficiency has greater impact on function of slow muscles, compared with fast, in aged BLAJ mice.

[This corrects the article DOI: 10.1371/journal.pone.0214908.].

Muscle Pathology in Dystrophic Rats and Zebrafish Is Unresponsive to Taurine Treatment, Compared to the mdx Mouse Model for Duchenne Muscular Dystrophy.

Inflammation and oxidative stress are strongly implicated in the pathology of Duchenne muscular dystrophy (DMD), and the sulphur-containing amino acid taurine ameliorates both and decreases dystropathology in the mdx mouse model for DMD. We therefore further tested taurine as a therapy using dystrophic DMDmdx rats and dmd zebrafish models for DMD that have a more severe dystropathology. However, taurine treatment had little effect on the indices of dystropathology in both these models. While we and others have previously observed a deficiency in taurine in mdx mice, in the current study we show that the rat and zebrafish models had increased taurine content compared with wild-type, and taurine treatment did not increase muscle taurine levels. We therefore hypothesised that endogenous levels of taurine are a key determinate in potential taurine treatment efficacy. Because of this, we felt it important to measure taurine levels in DMD patient plasma samples and showed that in non-ambulant patients (but not in younger patients) there was a deficiency of taurine. These data suggest that taurine homeostasis varies greatly between species and may be influenced by age and disease progression. The potential for taurine to be an effective therapy may depend on such variables.

Exercise builds the scaffold of life: muscle extracellular matrix biomarker responses to physical activity, inactivity, and aging.

Skeletal muscle extracellular matrix (ECM) is critical for muscle force production and the regulation of important physiological processes during growth, regeneration, and remodelling. ECM remodelling is a tightly orchestrated process, sensitive to multi-directional tensile and compressive stresses and damaging stimuli, and its assessment can convey important information on rehabilitation effectiveness, injury, and disease. Despite its profound importance, ECM biomarkers are underused in studies examining the effects of exercise, disuse, or aging on muscle function, growth, and structure. This review examines patterns of short- and long-term changes in the synthesis and concentrations of ECM markers in biofluids and tissues, which may be useful for describing the time course of ECM remodelling following physical activity and disuse. Forces imposed on the ECM during physical activity critically affect cell signalling while disuse causes non-optimal adaptations, including connective tissue proliferation. The goal of this review is to inform researchers, and rehabilitation, medical, and exercise practitioners better about the role of ECM biomarkers in research and clinical environments to accelerate the development of targeted physical activity treatments, improve ECM status assessment, and enhance function in aging, injury, and disease.

The location of protein oxidation in dystrophic skeletal muscle from the mdx mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe childhood disease characterised by progressive muscle wasting caused by widespread myofibre necrosis. Implicated in the pathology of DMD is oxidative stress, caused by excessive generation of reactive oxygen and nitrogen species (RONS). One consequence of RONS exposure is post-translational oxidative modifications to proteins, which can cause loss of protein function. This study used the dystrophic mdx mouse model for DMD to visualise the precise location of different oxidative modifications to proteins in dystrophic muscles, including both reversible (protein thiol oxidation and s-nitrosylation) and irreversible (carbonylation and dityrosine formation) oxidation at various stages of dystrophic muscle necrosis and regeneration. High levels of protein oxidation were observed in mdx myofibres undergoing degeneration and immune cell infiltration (myonecrosis). Since irreversible protein oxidation, especially dityrosine formation, was only colocalised to areas of myonecrosis, we suggest that this specific measurement could be a useful biomarker of myonecrosis. To test this we quantified dityrosines in muscle homogenates; this analysis showed significantly higher levels of dityrosines in mdx (compared with control normal) mice aged 23 days, an age when acute onset of extensive myonecrosis occurs in mdx muscles. These results indicate a major localised role of immune cells in RONS generation in dystrophic muscle, and strongly support a role for protein oxidation in myonecrosis and associated dystropathology. Consequently, the measurement of protein oxidation (specifically dityrosines) in dystrophic muscles may be a useful biomarker for indirectly quantifying myonecrosis in research studies using mdx mice and other animal models for DMD.

Dysferlin Deficiency Results in Myofiber-Type Specific Differences in Abundances of Calcium-Handling and Glycogen Metabolism Proteins.

Dysferlinopathies are a clinically heterogeneous group of muscular dystrophies caused by a genetic deficiency of the membrane-associated protein dysferlin, which usually manifest post-growth in young adults. The disease is characterized by progressive skeletal muscle wasting in the limb-girdle and limbs, inflammation, accumulation of lipid droplets in slow-twitch myofibers and, in later stages, replacement of muscles by adipose tissue. Previously we reported myofiber-type specific differences in muscle contractile function of 10-month-old dysferlin-deficient BLAJ mice that could not be fully accounted for by altered myofiber-type composition. In order to further investigate these findings, we examined the impact of dysferlin deficiency on the abundance of calcium (Ca2+) handling and glucose/glycogen metabolism-related proteins in predominantly slow-twitch, oxidative soleus and fast-twitch, glycolytic extensor digitorum longus (EDL) muscles of 10-month-old wild-type (WT) C57BL/6J and dysferlin-deficient BLAJ male mice. Additionally, we compared the Ca2+ activation properties of isolated slow- and fast-twitch myofibers from 3-month-old WT and BLAJ male mice. Differences were observed for some Ca2+ handling and glucose/glycogen metabolism-related protein levels between BLAJ soleus and EDL muscles (compared with WT) that may contribute to the previously reported differences in function in these BLAJ muscles. Dysferlin deficiency did not impact glycogen content of whole muscles nor Ca2+ activation of the myofilaments, although soleus muscle from 10-month-old BLAJ mice had more glycogen than EDL muscles. These results demonstrate a further impact of dysferlin deficiency on proteins associated with excitation-contraction coupling and glycogen metabolism in skeletal muscles, potentially contributing to altered contractile function in dysferlinopathy.

Three-dimensional mechanical characterization of murine skeletal muscle using quantitative micro-elastography.

Skeletal muscle function is governed by both the mechanical and structural properties of its constituent tissues, which are both modified by disease. Characterizing the mechanical properties of skeletal muscle tissue at an intermediate scale, i.e., between that of cells and organs, can provide insight into diseases such as muscular dystrophies. In this study, we use quantitative micro-elastography (QME) to characterize the micro-scale elasticity of ex vivo murine skeletal muscle in three-dimensions in whole muscles. To address the challenge of achieving high QME image quality with samples featuring uneven surfaces and geometry, we encapsulate the muscles in transparent hydrogels with flat surfaces. Using this method, we study aging and disease in quadriceps tissue by comparing normal wild-type (C57BL/6J) mice with dysferlin-deficient BLAJ mice, a model for the muscular dystrophy dysferlinopathy, at 3, 10, and 24 months of age (sample size of three per group). We observe a 77% decrease in elasticity at 24 months in dysferlin-deficient quadriceps compared to wild-type quadriceps.

Cilia, Centrosomes and Skeletal Muscle.

Primary cilia are non-motile, cell cycle-associated organelles that can be found on most vertebrate cell types. Comprised of microtubule bundles organised into an axoneme and anchored by a mature centriole or basal body, primary cilia are dynamic signalling platforms that are intimately involved in cellular responses to their extracellular milieu. Defects in ciliogenesis or dysfunction in cilia signalling underlie a host of developmental disorders collectively referred to as ciliopathies, reinforcing important roles for cilia in human health. Whilst primary cilia have long been recognised to be present in striated muscle, their role in muscle is not well understood. However, recent studies indicate important contributions, particularly in skeletal muscle, that have to date remained underappreciated. Here, we explore recent revelations that the sensory and signalling functions of cilia on muscle progenitors regulate cell cycle progression, trigger differentiation and maintain a commitment to myogenesis. Cilia disassembly is initiated during myoblast fusion. However, the remnants of primary cilia persist in multi-nucleated myotubes, and we discuss their potential role in late-stage differentiation and myofiber formation. Reciprocal interactions between cilia and the extracellular matrix (ECM) microenvironment described for other tissues may also inform on parallel interactions in skeletal muscle. We also discuss emerging evidence that cilia on fibroblasts/fibro-adipogenic progenitors and myofibroblasts may influence cell fate in both a cell autonomous and non-autonomous manner with critical consequences for skeletal muscle ageing and repair in response to injury and disease. This review addresses the enigmatic but emerging role of primary cilia in satellite cells in myoblasts and myofibers during myogenesis, as well as the wider tissue microenvironment required for skeletal muscle formation and homeostasis.

A Blood Biomarker for Duchenne Muscular Dystrophy Shows That Oxidation State of Albumin Correlates with Protein Oxidation and Damage in Mdx Muscle.

Duchenne muscular dystrophy (DMD) is a severe X-linked muscle wasting disease with no cure. While the precise mechanisms of progressive dystropathology remain unclear, oxidative stress caused by excessive generation of oxidants is strongly implicated. Blood biomarkers that could track oxidant levels in tissues would be valuable to measure the effectiveness of clinical treatments for DMD; our research has focused on developing such biomarkers. One target of oxidants that has the potential to be harnessed as a clinical biomarker is the thiol side chain of cysteine 34 (Cys34) of the blood protein albumin. This study using the mdx mouse model of DMD shows that in plasma, albumin Cys34 undergoes thiol oxidation and these changes correlate with levels of protein thiol oxidation and damage of the dystrophic muscles. A comparison with the commonly used biomarker protein carbonylation, confirmed that albumin thiol oxidation is the more sensitive plasma biomarker of oxidative stress occurring in muscle tissue. We show that plasma albumin oxidation reflects muscle dystropathology, as increased after exercise and decreased after taurine treatment of mdx mice. These data support the use of albumin thiol oxidation as a blood biomarker of dystropathology to assist with advancing clinical development of therapies for DMD.

Ageing contributes to phenotype transition in a mouse model of periodic paralysis.

BACKGROUND: Periodic paralysis (PP) is a rare genetic disorder in which ion channel mutation causes episodic paralysis in association with hyper- or hypokalaemia. An unexplained but consistent feature of PP is that a phenotype transition occurs around the age of 40, in which the severity of potassium-induced muscle weakness declines but onset of fixed, progressive weakness is reported. This phenotype transition coincides with the age at which muscle mass and optimal motor function start to decline in healthy individuals. We sought to determine if the phenotype transition in PP is linked to the normal ageing phenotype transition and to explore the mechanisms involved. METHODS: A mouse model of hyperkalaemic PP was compared with wild-type littermates across a range of ages (13-104 weeks). Only male mice were used as penetrance is incomplete in females. We adapted the muscle velocity recovery cycle technique from humans to examine murine muscle excitability in vivo. We then examined changes in potassium-induced weakness or caffeine contracture force with age using ex vivo muscle tension testing. Muscles were further characterized by either Western blot, histology or energy charge measurement. For normally distributed data, a student's t-test (± Welch correction) or one- or two-way analysis of variance (ANOVA) was performed to determine significance. For data that were not normally distributed, Welch rank test, Mann Whitney U test or Kruskal-Wallis ANOVA was performed. When an ANOVA was significant (P < 0.05), post hoc Tukey testing was used. RESULTS: Both WT (P = 0.009) and PP (P = 0.007) muscles exhibit increased resistance to potassium-induced weakness with age. Our data suggest that healthy-old muscle develops mechanisms to maintain force despite sarcolemmal depolarization and sodium channel inactivation. In contrast, reduced caffeine contracture force (P = 0.00005), skeletal muscle energy charge (P = 0.004) and structural core pathology (P = 0.005) were specific to Draggen muscle, indicating that they are caused, or at least accelerated by, chronic genetic ion channel dysfunction. CONCLUSIONS: The phenotype transition with age is replicated in a mouse model of PP. Intrinsic muscle ageing protects against potassium-induced weakness in HyperPP mice. However, it also appears to accelerate impairment of sarcoplasmic reticulum calcium release, mitochondrial impairment and the development of core-like regions, suggesting acquired RyR1 dysfunction as the potential aetiology. This work provides a first description of mechanisms involved in phenotype transition with age in PP. It also demonstrates how studying phenotype transition with age in monogenic disease can yield novel insights into both disease physiology and the ageing process itself.

Oxidative damage to urinary proteins from the GRMD dog and mdx mouse as biomarkers of dystropathology in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal, X-chromosome linked muscle-wasting disease affecting about 1 in 3500-6000 boys worldwide. Myofibre necrosis and subsequent loss of muscle mass are due to several molecular sequelae, such as inflammation and oxidative stress. We have recently shown increased neutrophils, highly reactive oxidant hypochlorous acid (HOCl) generation by myeloperoxidase (MPO), and associated oxidative stress in muscle from the GRMD dog and mdx mouse models for DMD. These findings have led us to hypothesise that generation of HOCl by myeloperoxidase released from neutrophils has a significant role in dystropathology. Since access to muscle from DMD patients is limited, the aim of this study was to develop methods to study this pathway in urine. Using immunoblotting to measure markers of protein oxidation, we show increased labelling of proteins with antibodies to dinitrophenylhydrazine (DNP, oxidative damage) and DiBrY (halogenation by reactive oxidants from myeloperoxidase) in GRMD and mdx urine. A strong positive correlation was observed between DiBrY labelling in dog urine and muscle. A strong positive correlation was also observed when comparing DNP and DiBrY labelling (in muscle and urine) to markers of dystropathology (plasma creatine kinase) and neutrophil presence (muscle MPO). Our results indicate the presence of neutrophil mediated oxidative stress in both models, and suggest that urine is a suitable bio-fluid for the measurement of such biomarkers. These methods could be employed in future studies into the role of neutrophil mediated oxidative stress in DMD and other inflammatory pathologies.

Dystrophic Dmdmdx rats show early neuronal changes (increased S100ß and Tau5) at 8 months, supporting severe dystropathology in this rodent model of Duchenne muscular dystrophy.

The intrinsic necrosis of skeletal muscles in animal models of Duchenne muscular dystrophy (DMD) damages neuromuscular junctions (NMJs) with progressively altered NMJs associated with denervation and premature changes in dystrophic nerves. In the mdx mouse model of DMD, the proteins S100ß and Tau5 are significantly increased in sciatic nerves by 13 months (M) of age, far earlier (by 9 M) than in normal wildtype (WT) nerves. Since dystrophic Dmdmdx rats are reported to have a more severe dystropathology than mdx mice, we hypothesised that Dmdmdx rat nerves would show earlier neuronal changes compared with mdx nerves. We quantified levels of 8 proteins (by immunoblotting) in sciatic and radial nerves from young adult Dmdmdx rats (aged 8 M) and mdx mice (9 M), plus levels of 7 mRNAs (by qPCR) in rat nerves only. Sciatic nerves of 8 M Dmdmdx rats had more consistently increased levels of S100ß and Tau5 proteins, compared with 9 M mdx mice, supporting pronounced dystropathology in the rat model. There were no differences for mRNA levels, apart from higher gelsolin mRNA in Dmdmdx sciatic nerves. The pronounced protein changes in Dmdmdx nerves indicate a severe ongoing myonecrosis, and likely consequent myofibre denervation, for the dystrophic rat model. These data support increased neuronal proteins in dystrophic nerves as a novel pre-clinical readout of ongoing myonecrosis for DMD research. In older DMD boys, such progressive neuronal changes over many years are likely to contribute to loss of muscle function, and may complicate evaluation of late-onset clinical therapies.

Mouse models for muscular dystrophies: an overview.

Muscular dystrophies (MDs) encompass a wide variety of inherited disorders that are characterized by loss of muscle tissue associated with a progressive reduction in muscle function. With a cure lacking for MDs, preclinical developments of therapeutic approaches depend on well-characterized animal models that recapitulate the specific pathology in patients. The mouse is the most widely and extensively used model for MDs, and it has played a key role in our understanding of the molecular mechanisms underlying MD pathogenesis. This has enabled the development of therapeutic strategies. Owing to advancements in genetic engineering, a wide variety of mouse models are available for the majority of MDs. Here, we summarize the characteristics of the most commonly used mouse models for a subset of highly studied MDs, collated into a table. Together with references to key publications describing these models, this brief but detailed overview would be useful for those interested in, or working with, mouse models of MD.

Biomarkers for Duchenne muscular dystrophy: myonecrosis, inflammation and oxidative stress.

Duchenne muscular dystrophy (DMD) is a lethal, X-linked disease that causes severe loss of muscle mass and function in young children. Promising therapies for DMD are being developed, but the long lead times required when using clinical outcome measures are hindering progress. This progress would be facilitated by robust molecular biomarkers in biofluids, such as blood and urine, which could be used to monitor disease progression and severity, as well as to determine optimal drug dosing before a full clinical trial. Many candidate DMD biomarkers have been identified, but there have been few follow-up studies to validate them. This Review describes the promising biomarkers for dystrophic muscle that have been identified in muscle, mainly using animal models. We strongly focus on myonecrosis and the associated inflammation and oxidative stress in DMD muscle, as the lack of dystrophin causes repeated bouts of myonecrosis, which are the key events that initiate the resultant severe dystropathology. We discuss the early events of intrinsic myonecrosis, along with early regeneration in the context of histological and other measures that are used to quantify its incidence. Molecular biomarkers linked to the closely associated events of inflammation and oxidative damage are discussed, with a focus on research related to protein thiol oxidation and to neutrophils. We summarise data linked to myonecrosis in muscle, blood and urine of dystrophic animal species, and discuss the challenge of translating such biomarkers to the clinic for DMD patients, especially to enhance the success of clinical trials.

Implications of increased S100ß and Tau5 proteins in dystrophic nerves of two mdx mouse models for Duchenne muscular dystrophy.

This study investigates changes with respect to increasing protein levels in dystrophic nerves of two mdx mouse models of Duchenne muscular dystrophy (DMD). We propose that these nerve changes result from progressive ongoing damage to neuromuscular junctions (NMJs) due to repeated intrinsic bouts of necrosis in dystrophic muscles. We compared sciatic nerves from classic mdx mice aged 13, 15 and 18 months (M), with D2.mdx mice (on DBA2 background) aged 9 and 13 M, using immunoblotting to quantify levels of 7 proteins. The neuronal proteins S100ß and Tau5 were increased by 13 M in mdx nerves (compared with WT), indicating ongoing myonecrosis in this strain. In striking contrast there was no difference in levels of these neuronal proteins for D2.mdx and D2.WT sciatic nerves at 13 M, indicating reduced myonecrosis over this time in D2.mdx mice compared with mdx. These novel changes in mdx sciatic nerves by 13 M, suggest early denervation or neurodegeneration of dystrophic nerves that is likely irreversible and progressive. This neuronal readout of persistent myonecrosis may provide a useful new long-term biomarker for preclinical studies that aim to reduce myonecrosis, plus such neuronal changes present potential new drug targets to help maintain the function of DMD muscles.

Improving translatability of preclinical studies for neuromuscular disorders: lessons from the TREAT-NMD Advisory Committee for Therapeutics (TACT).

Clinical trials for rare neuromuscular diseases imply, among other investments, a high emotional burden for the whole disease community. Translation of data from preclinical studies to justify any clinical trial must be carefully pondered in order to minimize the risk of clinical trial withdrawal or failure. A rigorous distinction between proof-of-concept and preclinical efficacy studies using animal models is key to support the rationale of a clinical trial involving patients. This Review evaluates the experience accumulated by the TREAT-NMD Advisory Committee for Therapeutics, which provides detailed constructive feedback on clinical proposals for neuromuscular diseases submitted by researchers in both academia and industry, and emphasizes that a timely critical review of preclinical efficacy data from animal models, including biomarkers for specific diseases, combined with adherence to existing guidelines and standard protocols, can significantly help to de-risk clinical programs and prevent disappointments and costly engagement.