The Experts below are selected from a list of 207 Experts worldwide ranked by ideXlab platform
Vilberto Stocchi - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH ARTICLE Muscle Releases Alpha-Sarcoglycan Positive Extracellular Vesicles Carrying miRNAs in the Bloodstream
2016Co-Authors: Michele Guescini, Barbara Canonico, Francesco Lucertini, Serena Maggio, Giosué Annibalini, Elena Barbieri, Francesca Luchetti, Stefano Papa, Vilberto StocchiAbstract:In the past few years, skeletal muscle has emerged as an important secretory organ produc-ing soluble factors, called myokines, that exert either autocrine, paracrine or endocrine ef-fects. Moreover, recent studies have shown that muscle releases microRNAs into the bloodstream in response to physical exercise. These microRNAs affect target cells, such as hormones and cytokines. The mechanisms underlying microRNA secretion are poorly char-acterized at present. Here, we investigated whether muscle tissue releases extracellular vesicles (EVs), which carry microRNAs in the bloodstream under physiological conditions such as physical exercise. Using density gradient separation of plasma from sedentary and physically fit young men we found EVs positive for TSG101 and Alpha-Sarcoglycan (SGCA), and enriched for miR-206. Cytometric analysis showed that the SGCA+ EVs ac-count for 1–5 % of the total and that 60–65 % of these EVs were also positive for the exoso-mal marker CD81. Furthermore, the SGCA-immuno captured sub-population of EVs exhibited higher levels of the miR-206/miR16 ratio compared to total plasma EVs. Finally, a significant positive correlation was found between the aerobic fitness and muscle-specific miRNAs and EV miR-133b and-181a-5p were significantly up-regulated after acute exer-cise. Thus, our study proposes EVs as a novel means of muscle communication potentially involved in muscle remodeling and homeostasis
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Muscle Releases Alpha-Sarcoglycan Positive Extracellular Vesicles Carrying miRNAs in the Bloodstream.
PloS one, 2015Co-Authors: Michele Guescini, Barbara Canonico, Francesco Lucertini, Serena Maggio, Giosué Annibalini, Elena Barbieri, Francesca Luchetti, Stefano Papa, Vilberto StocchiAbstract:In the past few years, skeletal muscle has emerged as an important secretory organ producing soluble factors, called myokines, that exert either autocrine, paracrine or endocrine effects. Moreover, recent studies have shown that muscle releases microRNAs into the bloodstream in response to physical exercise. These microRNAs affect target cells, such as hormones and cytokines. The mechanisms underlying microRNA secretion are poorly characterized at present. Here, we investigated whether muscle tissue releases extracellular vesicles (EVs), which carry microRNAs in the bloodstream under physiological conditions such as physical exercise. Using density gradient separation of plasma from sedentary and physically fit young men we found EVs positive for TSG101 and Alpha-Sarcoglycan (SGCA), and enriched for miR-206. Cytometric analysis showed that the SGCA+ EVs account for 1–5% of the total and that 60–65% of these EVs were also positive for the exosomal marker CD81. Furthermore, the SGCA-immuno captured sub-population of EVs exhibited higher levels of the miR-206/miR16 ratio compared to total plasma EVs. Finally, a significant positive correlation was found between the aerobic fitness and muscle-specific miRNAs and EV miR-133b and -181a-5p were significantly up-regulated after acute exercise. Thus, our study proposes EVs as a novel means of muscle communication potentially involved in muscle remodeling and homeostasis.
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Isolation of Alpha-Sarcoglycan+ EVs from plasma using immunoaffinity capturing.
2015Co-Authors: Michele Guescini, Barbara Canonico, Francesco Lucertini, Serena Maggio, Giosué Annibalini, Elena Barbieri, Francesca Luchetti, Stefano Papa, Vilberto StocchiAbstract:Anti-Alpha-Sarcoglycan antibodies were conjugated to magnetic beads to isolate muscle EVs from plasma. Western blot analysis confirmed the presence of the exosomal marker Tsg101 in isolated EVs. Ponceau S Staining has been used as loading control (a). MicroRNA quantifications showed an increase in the miR-206/miR-16 ratio in the SGCA+ sub-population of EVs (SGCA-Beads) compared to total (total plasma EVs) or uncaptured EVs (Supernatant). MiR-206 expression levels were normalized versus the endogenous reference miR-16 and expressed as -ΔCq (where ΔCq = CqmiR-206-CqmiR-16) (b). Moreover, the quantification of miR-16 ratio in the SGCA+ sub-population of EVs compared to total or uncaptured EVs shows that SGCA-conjugated beads retained about 2–5% of the total amount of EVs, miR-16 expression levels were normalized versus the spike-in reference cel-miR-39 and expressed as -ΔCq (where ΔCq = CqmiR-16-Cqcel-miR-39) (c). Asterisks denote significant changes (p
Jerry R Mendell - One of the best experts on this subject based on the ideXlab platform.
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RAPID COMMUNICATION Sustained Alpha-Sarcoglycan Gene Expression after Gene Transfer in Limb- Girdle Muscular Dystrophy, Type 2D
2015Co-Authors: Jerry R Mendell, Xiomara Q Rosales, Louise R. Rodino-klapac, Janaiah Kota, Reed K. ClarkAbstract:Objective: The aim of this study was to attain long-lasting Alpha-Sarcoglycan gene expression in limb-girdle muscular dystrophy, type 2D (LGMD2D) subjects mediated by adeno-associated virus (AAV) gene transfer under control of a muscle specific promoter (tMCK). Methods: rAAV1.tMCK.hSGCA (3.25 1011 vector genomes) was delivered to the extensor digitorum brevis muscle of 3 subjects with documented SGCA mutations via a double-blind, randomized, placebo controlled trial. Control sides received saline. The blind was not broken until the study was completed at 6 months and all results were reported to the oversight committee. Results: Persistent Alpha-Sarcoglycan gene expression was achieved for 6 months in 2 of 3 LGMD2D subjects. Markers for muscle fiber transduction other than Alpha-Sarcoglycan included expression of major histocompatibility complex I, increase in muscle fiber size, and restoration of the full Sarcoglycan complex. Mononuclear inflammatory cells recruited to the site of gene transfer appeared to undergo programmed cell death, demonstrated by terminal deoxynucleotide transferase–mediated deoxyuridine triphosphate nick-end labeling and caspase-3 staining. A patient failing gene transfer demonstrated an early rise in neutralizing antibody titers and T-cell immunity to AAV, validated by enzyme-linked immunospot on the second day after gene injection. This was in clear distinction to other participants with satisfactory gene expression
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sustained Alpha Sarcoglycan gene expression after gene transfer in limb girdle muscular dystrophy type 2d
Annals of Neurology, 2010Co-Authors: Jerry R Mendell, Louise R Rodinoklapac, Xiomara Q Rosales, Brian D Coley, Gloria Galloway, Sarah Lewis, Vinod MalikAbstract:Objective: The aim of this study was to attain long-lasting Alpha-Sarcoglycan gene expression in limb-girdle muscular dystrophy, type 2D (LGMD2D) subjects mediated by adeno-associated virus (AAV) gene transfer under control of a muscle specific promoter (tMCK). Methods: rAAV1.tMCK.hSGCA (3.25 � 10 11 vector genomes) was delivered to the extensor digitorum brevis muscle of 3 subjects with documented SGCA mutations via a double-blind, randomized, placebo controlled trial. Control sides received saline. The blind was not broken until the study was completed at 6 months and all results were reported to the oversight committee. Results: Persistent Alpha-Sarcoglycan gene expression was achieved for 6 months in 2 of 3 LGMD2D subjects. Markers for muscle fiber transduction other than Alpha-Sarcoglycan included expression of major histocompatibility complex I, increase in muscle fiber size, and restoration of the full Sarcoglycan complex. Mononuclear inflammatory cells recruited to the site of gene transfer appeared to undergo programmed cell death, demonstrated by terminal deoxynucleotide transferase–mediated deoxyuridine triphosphate nick-end labeling and caspase-3 staining. A patient failing gene transfer demonstrated an early rise in neutralizing antibody titers and T-cell immunity to AAV, validated by enzyme-linked immunospot on the second day after gene injection. This was in clear distinction to other participants with satisfactory gene expression. Interpretation: The findings of this gene replacement study in LGMD2D subjects have important implications not previously demonstrated in muscular dystrophy. Long-term, sustainable gene expression of Alpha-Sarcoglycan was observed following gene transfer mediated by AAV. The merit of a muscle-specific tMCK promoter, not previously used in a clinical trial, was evident, and the potential for reversal of disease was displayed. ANN NEUROL 2010;68:629–638
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lack of toxicity of Alpha Sarcoglycan overexpression supports clinical gene transfer trial in lgmd2d
Neurology, 2008Co-Authors: Louise R Rodinoklapac, J S Lee, Richard C Mulligan, K R Clark, Jerry R MendellAbstract:Background: Alpha-Sarcoglycan (α-SG) deficiency (limb-girdle muscular dystrophy [LGMD] type 2D) is the most common form of Sarcoglycan-LGMD. No treatment is currently available. Prior studies suggest that overexpression of α-SG via adeno-associated virus (AAV)-mediated gene transfer results in poorly sustained gene expression related to transgene toxicity. These findings potentially preclude gene therapy as a treatment approach for LGMD2D. Methods: The human α-SG gene (hα-SG) was directly transferred to the tibialis anterior muscle of 4- to 5-week-old α-SG KO mice using AAV, type 1. The gene was placed under control of either the ubiquitously expressed cytomegalovirus (CMV) promoter or muscle specific promoters that included desmin, muscle creatine kinase (MCK), and its further modification, truncated MCK (tMCK). Low (3 × 10 9 vg) and high (3 × 10 10 vg) doses of AAV1.hα-SG were administered. Results: Sustained gene expression was observed irrespective of promoters at 6 and 12 weeks post gene transfer. Quantitation of α-SG gene expression by fiber counts yielded similar levels of myofiber transduction for both MCK promoters (60 to 70%), while 34% of fibers were transduced with the DES promoter. There was a trend toward lower expression at the 12-week time point with the CMV promoter. Western blot analysis revealed α-SG overexpression using CMV and both the MCK promoters. Conclusion: Our data demonstrate robust and sustained adeno-associated virus type 1 Alpha-Sarcoglycan gene expression under control of muscle creatine kinase promoters, without evidence of cytotoxicity. These findings support the use of gene therapy as a potential treatment approach for limb-girdle muscular dystrophy type 2D.
Michele Guescini - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH ARTICLE Muscle Releases Alpha-Sarcoglycan Positive Extracellular Vesicles Carrying miRNAs in the Bloodstream
2016Co-Authors: Michele Guescini, Barbara Canonico, Francesco Lucertini, Serena Maggio, Giosué Annibalini, Elena Barbieri, Francesca Luchetti, Stefano Papa, Vilberto StocchiAbstract:In the past few years, skeletal muscle has emerged as an important secretory organ produc-ing soluble factors, called myokines, that exert either autocrine, paracrine or endocrine ef-fects. Moreover, recent studies have shown that muscle releases microRNAs into the bloodstream in response to physical exercise. These microRNAs affect target cells, such as hormones and cytokines. The mechanisms underlying microRNA secretion are poorly char-acterized at present. Here, we investigated whether muscle tissue releases extracellular vesicles (EVs), which carry microRNAs in the bloodstream under physiological conditions such as physical exercise. Using density gradient separation of plasma from sedentary and physically fit young men we found EVs positive for TSG101 and Alpha-Sarcoglycan (SGCA), and enriched for miR-206. Cytometric analysis showed that the SGCA+ EVs ac-count for 1–5 % of the total and that 60–65 % of these EVs were also positive for the exoso-mal marker CD81. Furthermore, the SGCA-immuno captured sub-population of EVs exhibited higher levels of the miR-206/miR16 ratio compared to total plasma EVs. Finally, a significant positive correlation was found between the aerobic fitness and muscle-specific miRNAs and EV miR-133b and-181a-5p were significantly up-regulated after acute exer-cise. Thus, our study proposes EVs as a novel means of muscle communication potentially involved in muscle remodeling and homeostasis
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Muscle Releases Alpha-Sarcoglycan Positive Extracellular Vesicles Carrying miRNAs in the Bloodstream.
PloS one, 2015Co-Authors: Michele Guescini, Barbara Canonico, Francesco Lucertini, Serena Maggio, Giosué Annibalini, Elena Barbieri, Francesca Luchetti, Stefano Papa, Vilberto StocchiAbstract:In the past few years, skeletal muscle has emerged as an important secretory organ producing soluble factors, called myokines, that exert either autocrine, paracrine or endocrine effects. Moreover, recent studies have shown that muscle releases microRNAs into the bloodstream in response to physical exercise. These microRNAs affect target cells, such as hormones and cytokines. The mechanisms underlying microRNA secretion are poorly characterized at present. Here, we investigated whether muscle tissue releases extracellular vesicles (EVs), which carry microRNAs in the bloodstream under physiological conditions such as physical exercise. Using density gradient separation of plasma from sedentary and physically fit young men we found EVs positive for TSG101 and Alpha-Sarcoglycan (SGCA), and enriched for miR-206. Cytometric analysis showed that the SGCA+ EVs account for 1–5% of the total and that 60–65% of these EVs were also positive for the exosomal marker CD81. Furthermore, the SGCA-immuno captured sub-population of EVs exhibited higher levels of the miR-206/miR16 ratio compared to total plasma EVs. Finally, a significant positive correlation was found between the aerobic fitness and muscle-specific miRNAs and EV miR-133b and -181a-5p were significantly up-regulated after acute exercise. Thus, our study proposes EVs as a novel means of muscle communication potentially involved in muscle remodeling and homeostasis.
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Isolation of Alpha-Sarcoglycan+ EVs from plasma using immunoaffinity capturing.
2015Co-Authors: Michele Guescini, Barbara Canonico, Francesco Lucertini, Serena Maggio, Giosué Annibalini, Elena Barbieri, Francesca Luchetti, Stefano Papa, Vilberto StocchiAbstract:Anti-Alpha-Sarcoglycan antibodies were conjugated to magnetic beads to isolate muscle EVs from plasma. Western blot analysis confirmed the presence of the exosomal marker Tsg101 in isolated EVs. Ponceau S Staining has been used as loading control (a). MicroRNA quantifications showed an increase in the miR-206/miR-16 ratio in the SGCA+ sub-population of EVs (SGCA-Beads) compared to total (total plasma EVs) or uncaptured EVs (Supernatant). MiR-206 expression levels were normalized versus the endogenous reference miR-16 and expressed as -ΔCq (where ΔCq = CqmiR-206-CqmiR-16) (b). Moreover, the quantification of miR-16 ratio in the SGCA+ sub-population of EVs compared to total or uncaptured EVs shows that SGCA-conjugated beads retained about 2–5% of the total amount of EVs, miR-16 expression levels were normalized versus the spike-in reference cel-miR-39 and expressed as -ΔCq (where ΔCq = CqmiR-16-Cqcel-miR-39) (c). Asterisks denote significant changes (p
Margot Jarrige - One of the best experts on this subject based on the ideXlab platform.
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Identification of thiostrepton as a pharmacological approach to rescue misfolded Alpha-Sarcoglycan mutant proteins from degradation
Scientific Reports, 2019Co-Authors: Lucile Hoch, Sara Henriques, Celine Bruge, Justine Marsolier, Manon Benabides, Nathalie Bourg, Johana Tournois, Gurvan Mahé, Lise Morizur, Margot JarrigeAbstract:Limb-girdle muscular dystrophy type 2D (LGMD2D) is characterized by a progressive proximal muscle weakness. LGMD2D is caused by mutations in the gene encoding α-Sarcoglycan (α-SG), a dystrophin-associated glycoprotein that plays a key role in the maintenance of sarcolemma integrity in striated muscles. We report here on the development of a new in vitro high-throughput screening assay that allows the monitoring of the proper localization of the most prevalent mutant form of α-SG (R77C substitution). Using this assay, we screened a library of 2560 FDA-approved drugs and bioactive compounds and identified thiostrepton, a cyclic antibiotic, as a potential drug to repurpose for LGMD2D treatment. Characterization of the thiostrepton effect revealed a positive impact on R77C-α-SG and other missense mutant protein localization (R34H, I124T, V247M) in fibroblasts overexpressing these proteins. Finally, further investigations of the molecular mechanisms of action of the compound revealed an inhibition of the chymotrypsin-like activity of the proteasome 24 h after thiostrepton treatment and a synergistic effect with bortezomib, an FDA-approved proteasome inhibitor. This study reports on the first in vitro model for LGMD2D that is compatible with high-throughput screening and proposes a new therapeutic option for LGMD2D caused by missense mutations of α-SG.
Louise R Rodinoklapac - One of the best experts on this subject based on the ideXlab platform.
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sustained Alpha Sarcoglycan gene expression after gene transfer in limb girdle muscular dystrophy type 2d
Annals of Neurology, 2010Co-Authors: Jerry R Mendell, Louise R Rodinoklapac, Xiomara Q Rosales, Brian D Coley, Gloria Galloway, Sarah Lewis, Vinod MalikAbstract:Objective: The aim of this study was to attain long-lasting Alpha-Sarcoglycan gene expression in limb-girdle muscular dystrophy, type 2D (LGMD2D) subjects mediated by adeno-associated virus (AAV) gene transfer under control of a muscle specific promoter (tMCK). Methods: rAAV1.tMCK.hSGCA (3.25 � 10 11 vector genomes) was delivered to the extensor digitorum brevis muscle of 3 subjects with documented SGCA mutations via a double-blind, randomized, placebo controlled trial. Control sides received saline. The blind was not broken until the study was completed at 6 months and all results were reported to the oversight committee. Results: Persistent Alpha-Sarcoglycan gene expression was achieved for 6 months in 2 of 3 LGMD2D subjects. Markers for muscle fiber transduction other than Alpha-Sarcoglycan included expression of major histocompatibility complex I, increase in muscle fiber size, and restoration of the full Sarcoglycan complex. Mononuclear inflammatory cells recruited to the site of gene transfer appeared to undergo programmed cell death, demonstrated by terminal deoxynucleotide transferase–mediated deoxyuridine triphosphate nick-end labeling and caspase-3 staining. A patient failing gene transfer demonstrated an early rise in neutralizing antibody titers and T-cell immunity to AAV, validated by enzyme-linked immunospot on the second day after gene injection. This was in clear distinction to other participants with satisfactory gene expression. Interpretation: The findings of this gene replacement study in LGMD2D subjects have important implications not previously demonstrated in muscular dystrophy. Long-term, sustainable gene expression of Alpha-Sarcoglycan was observed following gene transfer mediated by AAV. The merit of a muscle-specific tMCK promoter, not previously used in a clinical trial, was evident, and the potential for reversal of disease was displayed. ANN NEUROL 2010;68:629–638
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lack of toxicity of Alpha Sarcoglycan overexpression supports clinical gene transfer trial in lgmd2d
Neurology, 2008Co-Authors: Louise R Rodinoklapac, J S Lee, Richard C Mulligan, K R Clark, Jerry R MendellAbstract:Background: Alpha-Sarcoglycan (α-SG) deficiency (limb-girdle muscular dystrophy [LGMD] type 2D) is the most common form of Sarcoglycan-LGMD. No treatment is currently available. Prior studies suggest that overexpression of α-SG via adeno-associated virus (AAV)-mediated gene transfer results in poorly sustained gene expression related to transgene toxicity. These findings potentially preclude gene therapy as a treatment approach for LGMD2D. Methods: The human α-SG gene (hα-SG) was directly transferred to the tibialis anterior muscle of 4- to 5-week-old α-SG KO mice using AAV, type 1. The gene was placed under control of either the ubiquitously expressed cytomegalovirus (CMV) promoter or muscle specific promoters that included desmin, muscle creatine kinase (MCK), and its further modification, truncated MCK (tMCK). Low (3 × 10 9 vg) and high (3 × 10 10 vg) doses of AAV1.hα-SG were administered. Results: Sustained gene expression was observed irrespective of promoters at 6 and 12 weeks post gene transfer. Quantitation of α-SG gene expression by fiber counts yielded similar levels of myofiber transduction for both MCK promoters (60 to 70%), while 34% of fibers were transduced with the DES promoter. There was a trend toward lower expression at the 12-week time point with the CMV promoter. Western blot analysis revealed α-SG overexpression using CMV and both the MCK promoters. Conclusion: Our data demonstrate robust and sustained adeno-associated virus type 1 Alpha-Sarcoglycan gene expression under control of muscle creatine kinase promoters, without evidence of cytotoxicity. These findings support the use of gene therapy as a potential treatment approach for limb-girdle muscular dystrophy type 2D.