Proteasome inhibitors reduce thrombospondin-1 release in human dysferlin-deficient myotubes

Altres ajuts: This project has been funded by projects from the Fundación Isabel Gemio to II, EG and JDM and by Fundación Ramón Areces (CIVP18A3903) to NdL.Dysferlinopathies are a group of muscle disorders causing muscle weakness and absence or low levels of dysferlin, a type-II transmembrane protein and the causative gene of these dystrophies. Dysferlin is implicated in vesicle fusion, trafficking, and membrane repair. Muscle biopsy of patients with dysferlinopathy is characterized by the presence of inflammatory infiltrates. Studies in the muscle of both human and mouse models of dysferlinopathy suggest dysferlin deficient muscle plays a role in this inflammation by releasing thrombospondin-1. It has also been reported that vitamin D3 treatment enhances dysferlin expression. The ubiquitin-proteasome system recognizes and removes proteins that fail to fold or assemble properly and previous studies suggest that its inhibition could have a therapeutic effect in muscle dystrophies. Here we assessed whether inhibition of the ubiquitin proteasome system prevented degradation of dysferlin in immortalized myoblasts from a patients with two missense mutations in exon 44. To assess proteasome inhibition we treated dysferlin deficient myotubes with EB1089, a vitamin D3 analog, oprozomib and ixazomib. Western blot was performed to analyze the effect of these treatments on the recovery of dysferlin and myogenin expression. TSP-1 was quantified using the enzyme-linked immunosorbent assay to analyze the effect of these drugs on its release. A membrane repair assay was designed to assess the ability of treated myotubes to recover after membrane injury and fusion index was also measured with the different treatments. Data were analyzed using a one-way ANOVA test followed by Tukey post hoc test and analysis of variance. A p ≤ 0.05 was considered statistically significant. Treatment with proteasome inhibitors and EB1089 resulted in a trend towards an increase in dysferlin and myogenin expression. Furthermore, EB1089 and proteasome inhibitors reduced the release of TSP-1 in myotubes. However, no effect was observed on the repair of muscle membrane after injury. Our findings indicate that the ubiquitin-proteasome system might not be the main mechanism of mutant dysferlin degradation. However, its inhibition could help to improve muscle inflammation by reducing TSP-1 release. The online version contains supplementary material available at 10.1186/s12891-020-03756-7

Neuropsychopharmacology of Emerging Drugs of Abuse: meta- and para-Halogen-Ring-Substituted α-PVP (' flakka') Derivatives

Changes in the molecular structure of synthetic cathinones has led to an increase in the number of novel emerging drugs in the illicit drug market at an unprecedented rate. Unfortunately, little is known about the neuropsychopharmacology of recently emerged halogen-substituted -PVP derivatives. Thus, the aim of this study was to investigate the role of para- and meta-halogen (F-, Cl-, and Br-) substitutions on the in vitro, in silico, and in vivo effects of -pyrrolidinopentiophenone ( - PVP) derivatives. HEK293 cells expressing the human dopamine or serotonin transporter (hDAT and hSERT) were used for the uptake inhibition and transporter affinity assays. Molecular docking was used to model the interaction mechanism against DAT. Swiss CD-1 mice were used for the horizontal locomotor activity, open field test, and conditioned place preference paradigm. All compounds demonstrated potent DA uptake inhibition and higher DAT selectivity than cocaine. Meta-substituted cathinones showed higher DAT/SERT ratios than their para- analogs, which correlates with an increased psychostimulant effect in vivo and with different meta- and para-in silico interactions at DAT. Moreover, all compounds induced rewarding and acute anxiogenic effects in mice. In conclusion, the present study demonstrates the role of meta- and para-halogen substitutions in the mechanism of action and provides the first evidence of the rewarding and anxiety-like properties of halogenated -PVP derivatives

Nintedanib Reduces Muscle Fibrosis and Improves Muscle Function of the Alpha-Sarcoglycan-Deficient Mice

Sarcoglycanopathies are a group of recessive limb-girdle muscular dystrophies, characterized by progressive muscle weakness. Sarcoglycan deficiency produces instability of the sarcolemma during muscle contraction, leading to continuous muscle fiber injury eventually producing fiber loss and replacement by fibro-adipose tissue. Therapeutic strategies aiming to reduce fibro-adipose expansion could be effective in muscular dystrophies. We report the positive effect of nintedanib in a murine model of alpha-sarcoglycanopathy. We treated 14 Sgca mice, six weeks old, with nintedanib 50 mg/kg every 12 h for 10 weeks and compared muscle function and histology with 14 Sgca mice treated with vehicle and six wild-type littermate mice. Muscle function was assessed using a treadmill and grip strength. A cardiac evaluation was performed by echocardiography and histological study. Structural analysis of the muscles, including a detailed study of the fibrotic and inflammatory processes, was performed using conventional staining and immunofluorescence. In addition, proteomics and transcriptomics studies were carried out. Nintedanib was well tolerated by the animals treated, although we observed weight loss. Sgca mice treated with nintedanib covered a longer distance on the treadmill, compared with non-treated Sgca mice, and showed higher strength in the grip test. Moreover, nintedanib improved the muscle architecture of treated mice, reducing the degenerative area and the fibrotic reaction that was associated with a reversion of the cytokine expression profile. Nintedanib improved muscle function and muscle architecture by reducing muscle fibrosis and degeneration and reverting the chronic inflammatory environment suggesting that it could be a useful therapy for patients with alpha-sarcoglycanopathy

Identification of serum microRNAs as potential biomarkers in Pompe disease

Altres ajuts: This study was supported by a grant from Sanofi-Genzyme (GZ-2015-11342) to Dr. Gallardo and has been registered in Clinicaltrials.gov (identifier NCT03045042).This study was supported by a grant from Sanofi-Genzyme (GZ-2015-11342) to Dr. Gallardo and has been registered in Clinicaltrials.gov (identifier NCT03045042).To analyze the microRNA profile in serum of patients with Adult Onset Pompe disease (AOPD). We analyzed the expression of 185 microRNAs in serum of 15 AOPD patients and five controls using microRNA PCR Panels. The expression levels of microRNAs that were deregulated were further studied in 35 AOPD patients and 10 controls using Real-Time PCR. Additionally, the skeletal muscle expression of microRNAs which showed significant increase levels in serum samples was also studied. Correlations between microRNA serum levels and muscle function test, spirometry, and quantitative muscle MRI were performed (these data correspond to the study NCT01914536 at ClinicalTrials.gov). We identified 14 microRNAs that showed different expression levels in serum samples of AOPD patients compared to controls. We validated these results in a larger cohort of patients and we found increased levels of three microRNAs, the so called dystromirs: miR-1-3p, miR-133a-3p, and miR-206. These microRNAs are involved in muscle regeneration and the expression of these was increased in patients' muscle biopsies. Significant correlations between microRNA levels and muscle function test were found. Serum expression levels of dystromirs may represent additional biomarkers for the follow-up of AOPD patients

Antibodies against the flotillin-1/2 complex in patients with multiple sclerosis

Lleixa and Caballero-avila et al. report that antibodies targeting the flotillin-1/2 complex are present in a subgroup of patients with multiple sclerosis. Further studies are needed to understand the clinical and pathological relevance of anti-flotillin-1/2 autoantibodies in multiple sclerosis. Multiple sclerosis is a tissue-specific autoimmune disease of the central nervous system in which the antigen(s) remains elusive. Antibodies targeting the flotillin-1/2 complex have been described in 1-2% of the patients in a recent study. Other candidate antigens as anoctamin-2 or neurofascin-155 have been previously described in multiple sclerosis patients, although their clinical relevance remains uncertain. Our study aims to analyse the frequency and clinical relevance of antibodies against neurofascin-155, anoctamin-2 and flotillin-1/2 complex in multiple sclerosis. Serum (n = 252) and CSF (n = 50) samples from 282 multiple sclerosis patients were included in the study. The control group was composed of 260 serum samples (71 healthy donors and 189 with other neuroinflammatory disorders). Anti-flotillin-1/2, anti-anoctamin-2 and anti-neurofascin-155 antibodies were tested by cell-based assays using transfected cells. We identified six multiple sclerosis patients with antibodies against the flotillin-1/2 complex (2.1%) and one multiple sclerosis patient with antibodies against anoctamin-2 (0.35%). All multiple sclerosis patients were negative for anti-neurofascin-155 antibodies. Three of the anti-flotillin-1/2 positive patients showed anti-flotillin-1/2 positivity in other serum samples extracted at different moments of their disease. Immunoglobulin G subclasses of anti-flotillin-1/2 antibodies were predominantly one and three. We confirm that antibodies targeting the flotillin-1/2 complex are present in a subgroup of patients with multiple sclerosis. Further studies are needed to understand the clinical and pathological relevance of anti-flotillin-1/2 autoantibodies in multiple sclerosis

Identification of serum microRNAs as potential biomarkers in Pompe disease

Coinvestigators – The Spanish Pompe Study Group: Miguel Angel Barba-Romero; Joseba Barcena; María Rosario Carzorla; Carlota Creus; Jaume Coll-Canti; Noemí de Luna; Manuel Díaz; Cristina Domínguez; Roberto Fernández Torrón; María José García Antelo; Josep María Grau; María Teresa Gómez Caravaca; Juan Carlos León Hernández; Adolfo López de Munain; Francisco Antonio Martinez-García; Yolanda Morgado; Antonio Moreno; Germán Morís; Miguel Angel Muñoz-Blanco; Andres Nascimento; Carmen Paradas; José Luis Parajua Pozo; Luis Querol; Arturo Robledo-Strauss; Ricard Rojas García, Ricard; Íñigo Rojas-Marcoso; José Antonio Salazar; Mercedes Usón[Objective] To analyze the microRNA profile in serum of patients with Adult Onset Pompe disease (AOPD). [Methods] We analyzed the expression of 185 microRNAs in serum of 15 AOPD patients and five controls using microRNA PCR Panels. The expression levels of microRNAs that were deregulated were further studied in 35 AOPD patients and 10 controls using Real‐Time PCR. Additionally, the skeletal muscle expression of microRNAs which showed significant increase levels in serum samples was also studied. Correlations between microRNA serum levels and muscle function test, spirometry, and quantitative muscle MRI were performed (these data correspond to the study NCT01914536 at ClinicalTrials.gov). [Results] We identified 14 microRNAs that showed different expression levels in serum samples of AOPD patients compared to controls. We validated these results in a larger cohort of patients and we found increased levels of three microRNAs, the so called dystromirs: miR‐1‐3p, miR‐133a‐3p, and miR‐206. These microRNAs are involved in muscle regeneration and the expression of these was increased in patients' muscle biopsies. Significant correlations between microRNA levels and muscle function test were found. [Interpretation] Serum expression levels of dystromirs may represent additional biomarkers for the follow‐up of AOPD patients.This study was supported by a grant from Sanofi‐Genzyme (GZ‐2015‐11342) to Dr. Gallardo and has been registered in Clinicaltrials.gov (identifier NCT03045042)

Influence of Pain and Discomfort in Stroke Patients on Coping Strategies and Changes in Behavior and Lifestyle

The implementation of prevention strategies can reduce the risk of having a stroke. This prospective, longitudinal, multicenter observational study of 82 patients describes health habits, quality of life, coping strategies, and physical and neurological status at 3 months and 1 year after stroke. The EuroQoL-5D quality of life scale (EQ-5D) and the coping strategy measurement scale (COPE-28) were used to assess pain and discomfort, and behavioral and lifestyle changes. Significant differences were observed in the pain or discomfort levels of those patients with behavioral and lifestyle changes. Correlation was also found between pain or discomfort and the coping strategies associated with active emotional support at 1 year after stroke. The results of the pain or discomfort dimension were not, however, associated with better adherence to treatment. Pain and discomfort could have a predictive value in changes in lifestyles and behaviors but not for treatment adherence in patients who have had a stroke, which is significant at 1 year. In addition to important active coping strategies such as social support, these changes in behavior and lifestyle following a stroke are long-term and should therefore be assessed during the initial examination

Identification of serum microRNAs as potential biomarkers in Pompe disease

Altres ajuts: This study was supported by a grant from Sanofi-Genzyme (GZ-2015-11342) to Dr. Gallardo and has been registered in Clinicaltrials.gov (identifier NCT03045042).To analyze the microRNA profile in serum of patients with Adult Onset Pompe disease (AOPD). We analyzed the expression of 185 microRNAs in serum of 15 AOPD patients and five controls using microRNA PCR Panels. The expression levels of microRNAs that were deregulated were further studied in 35 AOPD patients and 10 controls using Real-Time PCR. Additionally, the skeletal muscle expression of microRNAs which showed significant increase levels in serum samples was also studied. Correlations between microRNA serum levels and muscle function test, spirometry, and quantitative muscle MRI were performed (these data correspond to the study NCT01914536 at ClinicalTrials.gov). We identified 14 microRNAs that showed different expression levels in serum samples of AOPD patients compared to controls. We validated these results in a larger cohort of patients and we found increased levels of three microRNAs, the so called dystromirs: miR-1-3p, miR-133a-3p, and miR-206. These microRNAs are involved in muscle regeneration and the expression of these was increased in patients' muscle biopsies. Significant correlations between microRNA levels and muscle function test were found. Serum expression levels of dystromirs may represent additional biomarkers for the follow-up of AOPD patients