Utrophin modulator drugs as potential therapies for Duchenne and Becker muscular dystrophies

Utrophin is an autosomal paralogue of dystrophin, a protein whose deficit causes Duchenne and Becker muscular dystrophies (DMD/BMD). Utrophin is naturally overexpressed at the sarcolemma of mature dystrophin-deficient fibres in DMD and BMD patients as well as in the mdx Duchenne mouse model. Dystrophin and utrophin can co-localise in human foetal muscle, in the dystrophin-competent fibres from DMD/BMD carriers, and revertant fibre clusters in biopsies from DMD patients. These findings suggest that utrophin overexpression could act as a surrogate, compensating for the lack of dystrophin, and, as such, it could be used in combination with dystrophin restoration therapies. Different strategies to overexpress utrophin are currently under investigation. In recent years, many compounds have been reported to modulate utrophin expression efficiently in preclinical studies and ameliorate the dystrophic phenotype in animal models of the disease. In this manuscript, we discuss the current knowledge on utrophin protein and the different mechanisms that modulate its expression in skeletal muscle. We also include a comprehensive review of compounds proposed as utrophin regulators and, as such, potential therapeutic candidates for these muscular dystrophies.This work was supported by funding from Health Institute Carlos III (ISCIII, Spain) and the European Regional Development Fund, (ERDF/FEDER), `A way of making Europe': Grant PI15/00333; Basque Government (grants 2016111029, 2018222035 and 2020333012) and Duchenne Parent Project Spain (grant 05/2016). P. S--M holds a Rio Hortega Fellowship from ISCIII (CM19/00104). V.A--G holds a Miguel Servet Fellowship from the ISCIII (CPII17/00004), part-funded by ERDF/FEDER. A. L--M acknowledges funding by Biocruces Bizkaia Health Research Institute (BC/I/DIV/19/001). V. A--G also acknowledges funding from Ikerbasque (Basque Foundation for Science). None of this funding represents a conflict of interest with the content of this review

A Novel Functional In Vitro Model that Recapitulates Human Muscle Disorders

Here, we aim to address the increasing need for a suitable human muscle in vitro model in order to advance in the knowledge of muscle pathophysiology and test novel therapies for muscle disorders. Our model is based on a simple 2D culture method that yields highly mature human myotubes under optimized environmental conditions. Culture conditions that produced functional and contractile human myotubes with an extended lifetime consisted in extracellular matrix overlay and addition of several trophic factors to the differentiation medium. In this work, we describe the generation of suitable models of muscular dystrophies (limb-girdle muscular dystrophy type 2A—LGMD2A and Duchenne) by silencing expression of key proteins in these myotubes. Western blot and immunocytochemical analyses demonstrated similar features between our knockdown human myotubes and dystrophic muscles in vivo, which support the general validity of our cellular models. We also found that both dystrophic models present higher resting cytosolic Ca2+ levels than controls, which support a common underlying deficit in calcium homeostasis. This novel human in vitro system would allow for high-throughput screening of new treatments for these muscular dystrophies as well as for other neuromuscular disorders. In addition, our model could be used to advance in our understanding of human skeletal muscle pathophysiology

Targeting the Ubiquitin-Proteasome System in Limb-Girdle Muscular Dystrophy With CAPN3 Mutations

[EN] LGMDR1 is caused by mutations in the CAPN3 gene that encodes calpain 3 (CAPN3), a non-lysosomal cysteine protease necessary for proper muscle function. Our previous findings show that CAPN3 deficiency leads to reduced SERCA levels through increased protein degradation. This work investigates the potential contribution of the ubiquitin-proteasome pathway to increased SERCA degradation in LGMDR1. Consistent with our previous results, we observed that CAPN3-deficient human myotubes exhibit reduced SERCA protein levels and high cytosolic calcium concentration. Treatment with the proteasome inhibitor bortezomib (Velcade) increased SERCA2 protein levels and normalized intracellular calcium levels in CAPN3-deficient myotubes. Moreover, bortezomib was able to recover mutated CAPN3 protein in a patient carrying R289W and R546L missense mutations. We found that CAPN3 knockout mice (C3KO) presented SERCA deficits in skeletal muscle in the early stages of the disease, prior to the manifestation of muscle deficits. However, treatment with bortezomib (0.8 mg/kg every 72 h) for 3 weeks did not rescue SERCA levels. No change in muscle proteasome activity was observed in bortezomib-treated animals, suggesting that higher bortezomib doses are needed to rescue SERCA levels in this model. Overall, our results lay the foundation for exploring inhibition of the ubiquitin-proteasome as a new therapeutic target to treat LGMDR1 patients. Moreover, patients carrying missense mutations in CAPN3 and presumably other genes may benefit from proteasome inhibition by rescuing mutant protein levels. Further studies in suitable models will be necessary to demonstrate the therapeutic efficacy of proteasome inhibition for different missense mutations.This research was funded by Diputación Foral de Gipuzkoa (AV-I, 2018-000117-01-B, 2019-00362-01-B); Fundación Gangoiti Barrera, Ministerio de Ciencia e Innovación (AV-I,PID 2020-119780RB-I00), the Basque Government (AV-I, ETORTEK-KK-2019/00093); the University of the Basque Country (AV-I, GIU20/057), and Instituto de Salud Carlos III, co-funded by European Regional Development Fund/ European Social Fund, “Investing in your future” (AV-I, PI17/00676; AL, PI17/01841). JL-E held a PhD fellowship from the Basque Government, LM-M holds a PhD fellowship from the UPV/EHU and GG holds a Juan de la Cierva- Incorporación 2019 contract funded by the Spanish Ministry of Science and Innovation

Electroactive 3D printable poly (3,4-ethylenedioxythiophene)-graft-poly(ε-caprolactone) copolymers as scaffolds for muscle cell alignment

Unformatted postprintThe development of tailor-made polymers to build artificial three-dimensional scaffolds to repair damaged skin tissues is gaining increasing attention in the bioelectronics field. Poly (3,4-ethylene dioxythiophene) (PEDOT) is the gold standard conducting polymer for the bioelectronics field due to its high conductivity, thermal stability, and biocompatibility; however, it is insoluble and infusible, which limits its processability into three dimensional scaffolds. Here, poly(3,4-ethylendioxythiophene)-graft-poly(ε−caprolactone) copolymers, PEDOT-g-PCL, with different molecular weights and PEDOT compositions, were synthesized by chemical oxidative polymerization to enhance the processability of PEDOT. First, the chemical structure and composition of the copolymers were characterized by nuclear magnetic resonance, infrared spectroscopy, and thermogravimetric analysis. Then, the additive manufacturing of PEDOT-g-PCL copolymers by direct ink writing was evaluated by rheology and 3D printing assays. The morphology of the printed patterns was further characterized by scanning electron microscopy and the conductivity by the four-point probe. Finally, the employment of these printed patterns to induce muscle cells alignment was tested, proving the ability of PEDOT-g-PCL patterns to produce myotubes differentiation.This work was funded by the spanish AEI-MICINN project PID2020-119026GB-I00 and Basque Government through grant IT1309-19. J. L. O.-M. thanks the Consejo Nacional de Ciencia y Tecnología (CONACyT, México) for the grant awarded no. 471837

Neural-Competent Cells of Adult Human Dermis Belong to the Schwann Lineage

SummaryResident neural precursor cells (NPCs) have been reported for a number of adult tissues. Understanding their physiological function or, alternatively, their activation after tissue damage or in vitro manipulation remains an unsolved issue. Here, we investigated the source of human dermal NPCs in adult tissue. By following an unbiased, comprehensive approach employing cell-surface marker screening, cell separation, transcriptomic characterization, and in vivo fate analyses, we found that p75NTR+ precursors of human foreskin can be ascribed to the Schwann (CD56+) and perivascular (CD56−) cell lineages. Moreover, neural differentiation potential was restricted to the p75NTR+CD56+ Schwann cells and mediated by SOX2 expression levels. Double-positive NPCs were similarly obtained from human cardiospheres, indicating that this phenomenon might be widespread

Deletion of integrin-linked kinase from neural crest cells in mice results in aortic aneurysms and embryonic lethality

SUMMARY Neural crest cells (NCCs) participate in the remodeling of the cardiac outflow tract and pharyngeal arch arteries during cardiovascular development. Integrin-linked kinase (ILK) is a serine/threonine kinase and a major regulator of integrin signaling. It links integrins to the actin cytoskeleton and recruits other adaptor molecules into a large complex to regulate actin dynamics and integrin function. Using the Cre-lox system, we deleted Ilk from NCCs of mice to investigate its role in NCC morphogenesis. The resulting mutants developed a severe aneurysmal arterial trunk that resulted in embryonic lethality during late gestation. Ilk mutants showed normal cardiac NCC migration but reduced differentiation into smooth muscle within the aortic arch arteries and the outflow tract. Within the conotruncal cushions, Ilk-deficient NCCs exhibited disorganization of F-actin stress fibers and a significantly rounder morphology, with shorter cellular projections. Additionally, absence of ILK resulted in reduced in vivo phosphorylation of Smad3 in NCCs, which correlated with reduced αSMA levels. Our findings resemble those seen in Pinch1 and β1 integrin conditional mutant mice, and therefore support that, in neural crest-derived cells, ILK and Pinch1 act as cytoplasmic effectors of β1 integrin in a pathway that protects against aneurysms. In addition, our conditional Ilk mutant mice might prove useful as a model to study aortic aneurysms caused by reduced Smad3 signaling, as occurs in the newly described aneurysms-osteoarthritis syndrome, for example

Increased expression of the astrocytic glutamate transporter GLT-1 in the prefrontal cortex of schizophrenics

glutamate uptake; astrocytes; schizophrenia; transporter regulation; synaptic transmission ABSTRACT To verify whether altered glial glutamate uptake contributes to the reduced efficacy of glutamatergic transmission reported in the prefrontal cortex of schizophrenics, we studied the expression of GLT-1, the transporter responsible for most glutamate transport, in autoptic samples of prefrontal cortex using real time quantitative RT-PCR, immunocytochemistry, and functional assays. GLT-1 mRNA levels in medication-free patients were 2.5-fold higher than in controls, whereas they were normal or reduced in patients treated with antipsychotics. We also observed a 4-fold increase in L- [ 3 H]-Glu uptake in Xenopus oocytes injected with mRNA from the prefrontal cortex of a medication-free schizophrenic and a 2-fold increase in GLT-1 protein in the same cortical area of another medication-free patient. Results suggest that GLT-1 mRNA, protein and function are increased in prefrontal cortex of schizophrenics. © 2004 Wiley-Liss, Inc. Following the demonstration that the psychotomimeti