Treatment with l-citrulline and metformin in Duchenne muscular dystrophy: study protocol for a single-centre, randomised, placebo-controlled trial

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Time-resolved pharmacological studies using automated, on-line monitoring of five parallel suspension cultures

Early stage pharmacological studies rely on in vitro methodologies for screening and testing compounds. Conventional assays based on endpoint measurements provide limited information because the lack in temporal resolution may not determine the pharmacological effect at its maximum. We developed an on-line, automated system for near real-time monitoring of extracellular content from five parallel suspension cultures, combining cell density measurements with a high-resolution separations every 12 minutes for 4 days. Selector and switching valves provide the fluidic control required to sample from one culture during the analysis of the previous sample from another culture, a time-saving measure that is fundamental to the throughput of the presented system. The system was applied to study the metabolic effects of the drugs rotenone, β-lapachone and clioquinol using lactate as metabolic indicator. For each drug, 96 assays were executed on the extracellular matrix at three concentrations with two controls in parallel, consuming only 5.78 mL of media from each culture over four days, less than 60 μL per analysis. The automated system provides high sample throughput, good temporal resolution and low sample consumption combined with a rugged analytical method with adequate sensitivity, providing a promising new platform for pharmacological and biotechnological studies

NQO1 protects against clioquinol toxicity

Clioquinol (CQ) was widely used as oral antibiotic before being taken off the market in many countries in 1970, after it was linked to subacute myelo-optic neuropathy (SMON) in Japan, leading to vision loss with many patients left wheelchair-bound. The common pathology of CQ-associated SMON was reproduced in animals but none of the proposed modes of toxicity explained the restriction of CQ-induced SMON to Japan. Given a re-emergence of CQ and related analogues as neuroprotectants, it is crucial to understand the underlying mechanism of CQ-induced toxicity to prevent any potential CQ-associated risks to future patients. A small molecule screen to find drugs that induce mitochondrial dysfunction in vitro identified CQ and the structurally related 8-hydroxyquinoline (8-OHQ). Their mitochondrial liability, pro-oxidative and cytotoxic activity was subsequently confirmed in some cell lines but surprisingly not in others. Subsequent studies in isogenic cell lines demonstrated that the antioxidant protein NQO1 is differentially expressed in the cell lines tested and potently protects against CQ toxicity. CQ-induced reduction of cellular ATP levels, increased lipid peroxidation and elevated cell death was also attenuated by antioxidants, implicating oxidative stress as the core mechanism of CQ-induced toxicity. These in-vitro findings were replicated in zebrafish. Visual acuity in zebrafish larvae that do not express NQO1, was reduced by CQ in a dose-dependent manner, while CQ did not affect visual function in the adult zebrafish that express NQO1. Similarly, pharmacological inhibition of NQO1 activity resulted in CQ-induced oxidative stress in the retina and severe acute systemic toxicity in the adult fish. Given the much higher prevalence of the inactivating C609T NQO1 polymorphism in the Japanese population compared to the European population, the results of this study could for the first time indicate how the geographic restriction of SMON cases to Japan could be explained. Importantly, if CQ or its derivatives are to be used safely for the treatment of neurodegenerative diseases, it seems imperative that NQO1 levels and activity of prospective patients should be ascertained

Adenylosuccinic acid therapy ameliorates murine Duchenne Muscular Dystrophy

International audienceArising from the ablation of the cytoskeletal protein dystrophin, Duchenne Muscular Dystrophy (DMD) is a debilitating and fatal skeletal muscle wasting disease underpinned by metabolic insufficiency. The inability to facilitate adequate energy production may impede calcium (Ca2+) buffering within, and the regenerative capacity of, dystrophic muscle. Therefore, increasing the metabogenic potential could represent an effective treatment avenue. The aim of our study was to determine the efficacy of adenylosuccinic acid (ASA), a purine nucleotide cycle metabolite, to stimulate metabolism and buffer skeletal muscle damage in the mdx mouse model of DMD. Dystrophin-positive control (C57BL/10) and dystrophin-deficient mdx mice were treated with ASA (3000 µg.mL-1) in drinking water. Following the 8-week treatment period, metabolism, mitochondrial density, viability and superoxide (O2-) production, as well as skeletal muscle histopathology, were assessed. ASA treatment significantly improved the histopathological features of murine DMD by reducing damage area, the number of centronucleated fibres, lipid accumulation, connective tissue infiltration and Ca2+ content of mdx tibialis anterior. These effects were independent of upregulated utrophin expression in the tibialis anterior. ASA treatment also increased mitochondrial viability in mdx flexor digitorum brevis fibres and concomitantly reduced O2- production, an effect that was also observed in cultured immortalised human DMD myoblasts. Our data indicates that ASA has a protective effect on mdx skeletal muscles

Targeting Nrf2 for the treatment of Duchenne Muscular Dystrophy

Imbalances in redox homeostasis can result in oxidative stress, which is implicated in various pathological conditions including the fatal neuromuscular disease Duchenne Muscular Dystrophy (DMD). DMD is a complicated disease, with many druggable targets at the cellular and molecular level including calcium-mediated muscle degeneration; mitochondrial dysfunction; oxidative stress; inflammation; insufficient muscle regeneration and dysregulated protein and organelle maintenance. Previous investigative therapeutics tended to isolate and focus on just one of these targets and, consequently, therapeutic activity has been limited. Nuclear erythroid 2-related factor 2 (Nrf2) is a transcription factor that upregulates many cytoprotective gene products in response to oxidants and other toxic stressors. Unlike other strategies, targeted Nrf2 activation has the potential to simultaneously modulate separate pathological features of DMD to amplify therapeutic benefits. Here, we review the literature providing theoretical context for targeting Nrf2 as a disease modifying treatment against DMD

Dimethyl Fumarate and Its Esters: A Drug with Broad Clinical Utility?

Fumaric acid esters (FAEs) are small molecules with anti-oxidative, anti-inflammatory and immune-modulating effects. Dimethyl fumarate (DMF) is the best characterised FAE and is approved and registered for the treatment of psoriasis and Relapsing-Remitting Multiple Sclerosis (RRMS). Psoriasis and RRMS share an immune-mediated aetiology, driven by severe inflammation and oxidative stress. DMF, as well as monomethyl fumarate and diroximel fumarate, are commonly prescribed first-line agents with favourable safety and efficacy profiles. The potential benefits of FAEs against other diseases that appear pathogenically different but share the pathologies of oxidative stress and inflammation are currently investigated

Standard of care versus new-wave corticosteroids in the treatment of Duchenne muscular dystrophy: Can we do better?

BACKGROUND: Pharmacological corticosteroid therapy is the standard of care in Duchenne Muscular Dystrophy (DMD) that aims to control symptoms and slow disease progression through potent anti-inflammatory action. However, a major concern is the significant adverse effects associated with long term-use. MAIN: This review discusses the pros and cons of standard of care treatment for DMD and compares it to novel data generated with the new-wave dissociative corticosteroid, vamorolone. The current status of experimental anti-inflammatory pharmaceuticals is also reviewed, with insights regarding alternative drugs that could provide therapeutic advantage. CONCLUSIONS: Although novel dissociative steroids may be superior substitutes to corticosteroids, other potential therapeutics should be explored. Repurposing or developing novel pharmacological therapies capable of addressing the many pathogenic features of DMD in addition to anti-inflammation could elicit greater therapeutic advantages

ΔNp63 transcriptionally regulates ATM to control p53 Serine-15 phosphorylation

Background: Delta Np63 alpha is an epithelial progenitor cell marker that maintains epidermal stem cell self-renewal capacity. Previous studies revealed that UV-damage induced p53 phosphorylation is confined to Delta Np63 alpha-positive cells in the basal layer of human epithelium. Results: We now report that phosphorylation of the p53 tumour suppressor is positively regulated by Delta Np63 alpha in immortalised human keratinocytes. Delta Np63 alpha depletion by RNAi reduces steady-state ATM mRNA and protein levels, and attenuates p53 Serine-15 phosphorylation. Conversely, ectopic expression of Delta Np63 alpha in p63-null tumour cells stimulates ATM transcription and p53 Serine-15 phosphorylation. We show that ATM is a direct Delta Np63 alpha transcriptional target and that the Delta Np63 alpha response element localizes to the ATM promoter CCAAT sequence. Structure-function analysis revealed that the Delta Np63-specific TA2 transactivation domain mediates ATM transcription in coordination with the DNA binding and SAM domains. Conclusions: Germline p63 point mutations are associated with a range of ectodermal developmental disorders, and targeted p63 deletion in the skin causes premature ageing. The Delta Np63 alpha-ATM-p53 damage-response pathway may therefore function in epithelial development, carcinogenesis and the ageing processes

Senataxin, defective in ataxia oculomotor apraxia type 2, is involved in the defense against oxidative DNA damage

Adefective response to DNA damage is observed in several human autosomal recessive ataxias with oculomotor apraxia, including ataxia-telangiectasia. We report that senataxin, defective in ataxia oculomotor apraxia (AOA) type 2, is a nuclear protein involved in the DNA damage response. AOA2 cells are sensitive to H2O2, camptothecin, and mitomycin C, but not to ionizing radiation, and sensitivity was rescued with full-length SETX cDNA. AOA2 cells exhibited constitutive oxidative DNA damage and enhanced chromosomal instability in response to H2O2. Rejoining of H2O2-induced DNA double-strand breaks (DSBs) was significantly reduced in AOA2 cells compared to controls, and there was no evidence for a defect in DNA single-strand break repair. This defect in DSB repair was corrected by full-length SETX cDNA. These results provide evidence that an additional member of the autosomal recessive AOA is also characterized by a defective response to DNA damage, which may contribute to the neurodegeneration seen in this syndrome

Functional link between BLM defective in Bloom's syndrome and the ataxia-telangiectasia-mutated protein, ATM

Chromosome aberrations, genomic instability, and cancer predisposition are hallmarks of a number of syndromes in which the defective genes recognize and/or repair DNA damage or are involved in some aspect of DNA processing. We report here direct interaction between BLM, mutated in Bloom's Syndrome (BS), and ATM, mutated is ataxia-telangiectasia, and we have mapped the sites of interaction. Full-length BLM cDNA corrected sister chromatid exchange (SCE) and radiosensitivity in BS cells. Mitotic phosphorylation of BLM was partially dependent on ATM, and phosphorylation sites on BLM were identified. A phosphospecific antibody against one of these sites (Thr-99) revealed radiation-induced phosphorylation, which was defective in ataxia-telangiectasia cells. Stable cell lines expressing phosphorylation site mutants failed to correct radiosensitivity in BS cells but corrected SCE. These mutants also sensitized normal control cells to radiation and increased radiation-induced chromosome aberrations but did not cause SCE numbers to increase. These data suggest that ATM and BLM function together in recognizing abnormal DNA structures by direct interaction and that these phosphorylation sites in BLM are important for radiosensitivity status but not for SCE frequency