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Background and objective: The determination of pharmacokinetic properties of new chemical entities is a key step in the process of drug development. Positron emission tomography (PET) is an ideal technique to obtain both biodistribution and pharmacokinetic parameters of new compounds over a wide range of chemical modalities. Here, we use a multi-radionuclide/multi-position labelling approach to investigate distribution, elimination, and metabolism of a triazole-based FKBP12 ligand (AHK2) with potential application in neuromuscular disorders. Methods: Target engagement and stabilizing capacity of the drug candidate (AHK2) towards FKBP12-RyR was evaluated using competitive ligand binding and proximity ligation assays, respectively. Subsequently, AHK2 was labelled either with the positron emitter carbon-11 (11C) via 11C-methylation to yield both [11C]AHK2.1 and [11C]AHK2.2, or by palladium-catalysed reduction of the corresponding 5-iodotriazole derivative using 3H gas to yield [3H]AHK2. Metabolism was first investigated in vitro using liver microsomes. PET imaging studies in rats after intravenous (IV) administration at different doses (1 µg/Kg and 5 mg/Kg) were combined with determination of arterial blood time-activity curves (TACs) and analysis of plasma samples by high performance liquid chromatography (HPLC) to quantify radioactive metabolites. Arterial TACs were obtained in continuous mode by using an in-house developed system that enables extracorporeal blood circulation and continuous measurement of radioactivity in the blood. Pharmacokinetic parameters were determined by non-compartmental modelling of the TACs. Results: In vitro studies indicate that AHK2 binds to FKBP12 at the rapamycin-binding pocket, presenting activity as a FKBP12/RyR stabilizer. [11C]AHK2.1, [11C]AHK2.2 and [3H]AHK2 could be obtained in overall non-decay corrected radiochemical yields of 14 ± 2%, 15 ± 2% and 0.05%, respectively. Molar activities were 60–110 GBq/µmol, 68–122 GBq/µmol and 0

Aged muscles accumulate satellite cells with a striking decline response to damage. Although intrinsic defects in satellite cells themselves are the major contributors to aging-associated stem cell dysfunction, increasing evidence suggests that changes in the muscle-stem cell local microenvironment also contribute to aging. Here, we demonstrate that loss of the matrix metalloproteinase-10 (MMP-10) in young mice alters the composition of the muscle extracellular matrix (ECM), and specifically disrupts the extracellular matrix of the satellite cell niche. This situation causes premature features of aging in the satellite cells, contributing to their functional decline and a predisposition to enter senescence under proliferative pressure. Similarly, reduction of MMP-10 levels in young satellite cells from wild type animals induces a senescence response, while addition of the protease delays this program. Significantly, the effect of MMP-10 on satellite cell aging can be extended to another context of muscle wasting, muscular dystrophy. Systemic treatment of mdx dystrophic mice with MMP-10 prevents the muscle deterioration phenotype and reduces cellular damage in the satellite cells, which are normally under replicative pressure. Most importantly, MMP-10 conserves its protective effect in the satellite cell-derived myoblasts isolated from a Duchenne muscular dystrophy patient by decreasing the accumulation of damaged DNA. Hence, MMP-10 provides a previously unrecognized therapeutic opportunity to delay satellite cell aging and overcome satellite cell dysfunction in dystrophic muscles.

[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.

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.

Páginas 114-122 confidenciales. Tesis completa174 p.-- Tesis censurada 167 p., Limb-girdle muscle dystrophy type R1 (LGMDR1) is the most common form of limb-girdle muscle dystrophy, currently with no effective treatment. LGMDR1 is caused by mutations in the CAPN3 gene and is characterized by reduced mobility, making daily live activities challenging. CAPN3 gene encodes calpain 3 protein (CAPN3), a non-lysosomal cysteine protease necessary for proper muscle function. Although the main function of CAPN3 in skeletal muscle and the pathophysiological mechanisms underlying the disease remain to be clarified, previous studies suggest that dysregulation of calcium (Ca2+) homeostasis is involved in the pathogenic mechanisms of this muscular dystrophy. In fact, our previous data suggest that CAPN3 deficiency may trigger calcium handling proteins dysfunction such as Sarco-Endoplasmic Reticulum ATPase pumps (SERCA) due to its over-ubiquitination and its consequent exacerbated degradation by Ubiquitin-Proteasome System (UPS). In the present work, we have characterized SERCA expression in different human in vitro LGMDR1 models observing that CAPN3 dysfunction generates SERCA protein downregulation, leading to increase basal intracellular calcium levels. Moreover, CAPN3-deficient LHCN-M2 human myotubes show upregulated sarcoplasmic reticulum (SR) stress markers. We have targeted SERCA degradation inhibiting UPS by Bortezomib (BTZ) treatment, which recovers not only SERCA2 protein expression but also mutated CAPN3 expression, restoring at least its structural function, and consequently, normalizing resting cytosolic Ca2+ levels. Despite the mild phenotype observed in C3KO mouse model, SERCA protein expression is reduced particularly in the diaphragm, as in C3 null rats, which show slightly more severe phenotype. However, due to the general modest phenotype of LGMDR1 animal models, the narrow therapeutic window between effectiveness and toxicity of BTZ treatment, and the high susceptibility of the rat model to Bortezomib, SERCA protein expression is not resto

Dystrophin-null sapje zebrafish is an excellent model for better understanding the pathological mechanisms underlying Duchenne muscular dystrophy, and it has recently arisen as a powerful tool for high-throughput screening of therapeutic candidates for this disease. While dystrophic phenotype in sapje larvae can be easily detected by birefringence, zebrafish genotyping is necessary for drug screening experiments, where the potential rescue of larvae phenotype is the primary outcome. Genotyping is also desirable during colony husbandry since heterozygous progenitors need to be selected. Currently, sapje zebrafish are genotyped through techniques involving sequencing or multi-step PCR, which are often costly, tedious, or require special equipment. Here we report a simple, precise, cost-effective, and versatile PCR genotyping method based on primer competition. Genotypes can be resolved by standard agarose gel electrophoresis and high-resolution melt assay, the latter being especially useful for genotyping a large number of samples. Our approach has shown high sensitivity, specificity, and reproducibility in detecting the A/T point mutation in sapje zebrafish and the C/T mutation in the mdx mouse model of Duchenne. Hence, this method can be applied to other single nucleotide substitutions and may be further optimized to detect small insertions and deletions. Given its robust performance with crude DNA extracts, our strategy may be particularly well-suited for detecting single nucleotide variants in poor-quality samples such as ancient DNA or DNA from formalin-fixed, paraffin-embedded material.

140 p., Duchenne muskulu-distrofia (DMD) gaixotasun neuromuskular larria da eta X kromosoman dagoen distrofinaren geneko mutazio azpirakorrak dira erantzuleak. 3500 mutiletik bati eragiten dio batez beste eta, gaur egun, sendabiderik ez duen eta heriotza goiztiarra dakarren gaitza da. DMDn gihar-eskeletikoa eta bihotza kaltetuta daude, ahultasun eta endekapen progresiboa eta, azken fasean, bihotzeko gutxiegitasuna eraginez; horrez gain, gaixoen %30ak nerbio-sistema zentralean ere arazoak ditu. Distrofina ez izateak zeluletako estres nitro-oxidatzailea areagotzen du eta hainbat proteinetan itzulpen-ondorengo aldaketak eragiten ditu. Besteak beste, kaltzio-kanal diren rianodina-hartzaileen (RyR) konformazioari eragiten die ezin baitzaie kalstabina behar bezala lotu eta kalstabina batuta ez dagoenean RyR ez da behar bezala itxiko kaltzio-jarioak eraginez.Lan honen helburua kalstabina proteina modulatuz RyR kaltzio-kanalen funtzioa berrezartzeko diseinatu diren AHK molekulen eraginkortasuna aztertzea izan da Duchenne muskulu-distrofiaren animalia-eredu diren sapje zebra-arrainetan eta mdx saguetan. Zebra-arrainetan AHK molekulek itu berarekin diseinatutako beste konposatu batzuk baino toxikotasun gutxiago dute, eta sapje arrainetako fenotipo distrofikoa aldatzen ez duten arren, AHK1ek eta AHK2k indar aktiboa emendatzen dute. AHK2a giharrera, bihotzera eta garunera heltzen dela baieztatu da mdx saguetan. Animalia-eredu honetan AHK2aren 12 asteko tratamenduaren ondoren hobekuntza nabarmenak ikusi dira bihotzaren funtzionamenduan eta saguei egindako kognizio probetan. Funtzio muskularrari dagokionez, aurrez 5 asteko tratamenduarekin maila funtzional eta histopatologikoan ikusitako hobekuntzak diluituago ageri dira 12 asteko tratamenduaren ondoren; honela, AHK2ak mdx saguen giharretan duen eraginkortasuna gaixotasunaren eboluzioarekin gutxitzen dela ebatzi da.

Limb-girdle muscular dystrophy recessive 1 (LGMDR1), previously known as LGMD2A, is a rare disease caused by mutations in the CAPN3 gene. It is characterized by progressive weakness of shoulder, pelvic, and proximal limb muscles that usually appears in children and young adults and results in loss of ambulation within 20 years after disease onset in most patients. The pathophysiological mechanisms involved in LGMDR1 remain mostly unknown, and to date, there is no effective treatment for this disease. Here, we review clinical and experimental evidence suggesting that dysregulation of Ca2+ homeostasis in the skeletal muscle is a significant underlying event in this muscular dystrophy. We also review and discuss specific clinical features of LGMDR1, CAPN3 functions, novel putative targets for therapeutic strategies, and current approaches aiming to treat LGMDR1. These novel approaches may be clinically relevant not only for LGMDR1 but also for other muscular dystrophies with secondary calpainopathy or with abnormal Ca2+ homeostasis, such as LGMD2B/LGMDR2 or sporadic inclusion body myositis.

157 p., La distrofia muscular de Duchenne (DMD) es una enfermedad hereditaria ligada al cromosoma X queafecta a uno de cada 3500 varones. Esta enfermedad se caracteriza por la degeneración progresiva de lasfibras musculares que conlleva debilidad muscular, incapacidad y muerte prematura.Desafortunadamente, actualmente no existe terapia eficaz para el tratamiento de estos pacientes. La DMDestá producida por mutaciones en el gen que codifica la distrofina una proteína de membrana que actúacomo amortiguador y a su vez sirve de anclaje para diversas proteínas de señalización que son esencialespara el correcto funcionamiento muscular. A su vez, se ha visto que en la DMD los receptores derianodina (RyR) sufren modificaciones post-transcripcionales que afectan su unión a la calstabina (Calst),y generan una pérdida de calcio desde el retículo sarcoplásmico, lo que conlleva al aumento de losniveles de calcio intracelulares. En el presente trabajo, los compuestos AHK que han sido diseñados paraactuar sobre esta prometedora diana terapéutica en colaboración con el grupo del Dr Aizpurua de laUniversidad del País Vasco, han demostrado aumentar significativamente la interacción RyR-Calstmejorando el fenotipo distrófico en ratones mdx, modelo animal de DMD. A su vez, el efecto de loscompuestos AHK ha sido testado in vitro en un modelo humano de DMD, donde han demostrado sueficacia aumentando la interacción RyR1-Calst1 y provocando la reducción de los niveles de calciointracelular. Por todo ello, se concluye que los compuestos AHK podrían consolidar una alternativaterapéutica de gran utilidad para el tratamiento de la DMD.

We have developed a simple and inexpensive method that improves sensitivity of protein and antigen detection in standard PAGE procedures. Our technique uses a sample microloader device with a funnel-like structure, filled with a 4% stacking gel. When attach to the top of a polyacrylamide slab gel, the proteins in a sample are concentrated by electrophoresis into a small volume as they emerge from the device's narrow outlet. Our microloader has several advantages over previous devices, including simple assembly, high versatility, and absence of cross-contamination between lanes. Addition of this device to a slab gel results in a fivefold increase in the sensitivity of antigen detection in a Western blot. As a result, less protein is required for loading and signal detection. Our protocol is a straightforward modification of a standard experimental technique, and is especially useful when only limited sample quantities are available.