The Experts below are selected from a list of 2409 Experts worldwide ranked by ideXlab platform
Elizabeth M. Mcnally - One of the best experts on this subject based on the ideXlab platform.
-
distinct pathophysiological mechanisms of cardiomyopathy in hearts lacking dystrophin or the Sarcoglycan complex
The FASEB Journal, 2011Co-Authors: Dewayne Townsend, Elizabeth M. Mcnally, Soichiro Yasuda, Joseph M MetzgerAbstract:Duchenne muscular dystrophy (DMD) and limb girdle muscular dystrophy (LGMD) 2C-F result from the loss of dystrophin and the Sarcoglycans, respectively. Dystrophin, a cytoskeletal protein, is closely associated with the membrane-bound Sarcoglycan complex. Despite this tight biochemical association, the function of dystrophin and the Sarcoglycan subunits may differ. The loss of dystrophin in skeletal muscle results in muscle that is highly susceptible to contraction-induced damage, but the skeletal muscle of mice lacking γ- or δ-Sarcoglycan are less susceptible. Using mouse models of DMD, LGMD-2C, and LGMD-2F, we demonstrate that isolated cardiac myocytes from mice lacking either γ- or δ-Sarcoglycan have normal compliance. In contrast, dystrophin-deficient myocytes display poor passive compliance and are susceptible to terminal contracture following mild passive extensions. Mice deficient in dystrophin and, less so, δ-Sarcoglycan have reduced survival during in vivo dobutamine stress testing compared to controls. Catheter-based hemodynamic studies show deficits in both baseline and dobutamine-stimulated cardiac function in all of the dystrophic mice compared to control mice, with dystrophin-deficient mice having the poorest function. In contrast, histopathology showed increased fibrosis in the Sarcoglycan-deficient hearts, but not in hearts lacking dystrophin. In summary, this study provides important insights into the unique mechanisms of disease underlying these different models of inherited dystrophic cardiomyopathy and supports a model where dystrophin, but not the Sarcoglycans, protects the cardiac myocyte against mechanical damage.—Townsend, D., Yasuda, S., McNally, E., Metzger, J. M. Distinct pathophysiological mechanisms of cardiomyopathy in hearts lacking dystrophin or the Sarcoglycan complex.
-
Reduced life span with heart and muscle dysfunction in Drosophila Sarcoglycan mutants.
Human molecular genetics, 2007Co-Authors: Michael J. Allikian, Gira Bhabha, Patrick Dospoy, Ahlke Heydemann, Pearl V. Ryder, Judy U. Earley, Matthew J. Wolf, Howard A. Rockman, Elizabeth M. McnallyAbstract:In humans, genetically diverse forms of muscular dystrophy are associated with a disrupted Sarcoglycan complex. The Sarcoglycan complex resides at the muscle plasma membrane where it associates with dystrophin. There are six known Sarcoglycan proteins in mammals whereas there are only three in Drosophila melanogaster. Using imprecise P element excision, we generated three different alleles at the Drosophila delta-Sarcoglycan locus. Each of these deletions encompassed progressively larger regions of the delta-Sarcoglycan gene. Line 840 contained a large deletion of the delta-Sarcoglycan gene, and this line displayed progressive impairment in locomotive ability, reduced heart tube function and a shortened life span. In line 840, deletion of the Drosophila delta-Sarcoglycan gene produced disrupted flight muscles with shortened sarcomeres and disorganized M lines. Unlike mammalian muscle where degeneration is coupled with ongoing regeneration, no evidence for regeneration was seen in this Drosophila Sarcoglycan mutant. In contrast, line 28 was characterized with a much smaller deletion that affected only a portion of the cytoplasmic region of the delta-Sarcoglycan protein and left intact the transmembrane and extracellular domains. Line 28 had a very mild phenotype with near normal life span, intact cardiac function and normal locomotive activity. Together, these data demonstrate the essential nature of the transmembrane and extracellular domains of Drosophila delta-Sarcoglycan for normal muscle structure and function.
-
nuclear sequestration of δ Sarcoglycan disrupts the nuclear localization of lamin a c and emerin in cardiomyocytes
Human Molecular Genetics, 2007Co-Authors: Ahlke Heydemann, Michele Hadhazy, Judy U. Earley, Alexis R Demonbreun, Elizabeth M. McnallyAbstract:Sarcoglycan is a membrane-associated protein complex found at the plasma membrane of cardiomyocytes and skeletal myofibers. Recessive mutations of d-Sarcoglycan that eliminate expression, and therefore function, lead to cardiomyopathy and muscular dystrophy by producing instability of the plasma membrane. A dominant missense mutation in the gene encoding d-Sarcoglycan was previously shown to associate with dilated cardiomyopathy in humans. To investigate the mechanism of dominantly inherited cardiomyopathy, we generated transgenic mice that express the S151A d-Sarcoglycan mutation in the heart using the a-myosin heavy-chain gene promoter. Similar to the human d-Sarcoglycan gene mutation, S151A d-Sarcoglycan transgenic mice developed dilated cardiomyopathy at a young age with enhanced lethality. Instead of placement at the plasma membrane, d-Sarcoglycan was found in the nucleus of S151A d-Sarcoglycan cardiomyocytes. Retention of d-Sarcoglycan in the nucleus was accompanied by partial nuclear sequestration of b -a ndg-Sarcoglycan. Additionally, the nuclear-membrane-associated proteins, lamin A/C and emerin, were mislocalized throughout the nucleoplasm. Therefore, the S151A d-Sarcoglycan gene mutation acts in a dominant negative manner to produce trafficking defects that disrupt nuclear localization of lamin A/C and emerin, thus linking together two common mechanisms of inherited cardiomyopathy.
-
genetic compensation for Sarcoglycan loss by integrin α7β1 in muscle
Journal of Cell Science, 2004Co-Authors: Michael J. Allikian, Andrew A. Hack, Stephanie K Mewborn, Ulrike Mayer, Elizabeth M. McnallyAbstract:Disruption of the Sarcoglycan complex leads to muscle membrane instability and muscular dystrophy in humans and mice. Through the dystrophin glycoprotein complex, Sarcoglycan participates in connecting the internal cytoskeleton to the membrane and the extracellular matrix. Integrin α7β1 is also a transmembrane protein of skeletal and cardiac muscle that similarly links the cytoskeleton to the extracellular matrix. Mice lacking integrin α7 develop mild muscle degeneration, while Sarcoglycan mutant mice display overt muscle degeneration and muscular dystrophy. In Sarcoglycan-deficient muscle, integrin α7 protein was upregulated at the plasma membrane. To ascertain whether integrin α7 upregulation compensates for the loss of the transmembrane Sarcoglycan linkage in Sarcoglycan-deficient muscle, we generated mice lacking both integrin α7 and γ-Sarcoglycan (gxi). These double-mutant gxi mice exhibit profound, rapid muscle degeneration leading to death before one month of age consistent with a weakened cellular attachment to the extracellular matrix. The regenerative capacity of gxi muscle was intact with increased embryonic myosin heavy chain expression, myofiber central nucleation and normal in vivo myoblast differentiation. Therefore, upregulation of integrin α7β1 compensates as a transmembrane muscle cell attachment for Sarcoglycan consistent with overlapping roles for Sarcoglycan and integrins in mediating cytoskeletal-membrane-extracellular matrix interaction.
-
smooth muscle cell extrinsic vascular spasm arises from cardiomyocyte degeneration in Sarcoglycan deficient cardiomyopathy
Journal of Clinical Investigation, 2004Co-Authors: Matthew T. Wheeler, Michele Hadhazy, Michael J. Allikian, Ahlke Heydemann, Sara Zarnegar, Elizabeth M. McnallyAbstract:Vascular spasm is a poorly understood but critical biomedical process because it can acutely reduce blood supply and tissue oxygenation. Cardiomyopathy in mice lacking γ-Sarcoglycan or δ-Sarcoglycan is characterized by focal damage. In the heart, Sarcoglycan gene mutations produce regional defects in membrane permeability and focal degeneration, and it was hypothesized that vascular spasm was responsible for this focal necrosis. Supporting this notion, vascular spasm was noted in coronary arteries, and disruption of the Sarcoglycan complex was observed in vascular smooth muscle providing a molecular mechanism for spasm. Using a transgene rescue strategy in the background of Sarcoglycan-null mice, we replaced cardiomyocyte Sarcoglycan expression. Cardiomyocyte-specific Sarcoglycan expression was sufficient to correct cardiac focal degeneration. Intriguingly, successful restoration of the cardiomyocyte Sarcoglycan complex also eliminated coronary artery vascular spasm, while restoration of smooth muscle Sarcoglycan in the background of Sarcoglycan-null alleles did not. This mechanism, whereby tissue damage leads to vascular spasm, can be partially corrected by NO synthase inhibitors. Therefore, we propose that cytokine release from damaged cardiomyocytes can feed back to produce vascular spasm. Moreover, vascular spasm feeds forward to produce additional cardiac damage.
Carsten G. Bönnemann - One of the best experts on this subject based on the ideXlab platform.
-
primary γ Sarcoglycanopathy lgmd 2c broadening of the mutational spectrum guided by the immunohistochemical profile
Neuromuscular Disorders, 2002Co-Authors: K J Jones, Carsten G. Bönnemann, Hart G.w. Lidov, Jenise C Wong, Chris A FeenerAbstract:Abstract An important step in the diagnostic evaluation of a patient with recessive limb-girdle muscular dystrophy is the immunohistochemical analysis of the components of the Sarcoglycan complex in a muscle biopsy specimen. Even though a primary mutation in any of the four Sarcoglycan genes (α-, β-,γ-, δ-Sarcoglycan) may cause secondary deficiencies in all the other Sarcoglycan proteins, more specific immunohistochemical patterns have emerged with the potential to guide and abbreviate the necessary molecular genetic investigations. In γ-Sarcoglycan mutations, the pattern consists of absent or prominently reduced γ-Sarcoglycan immunoreactivity in combination with reduced but detectable immunoreactivity for the other components, with preservation of δ-Sarcoglycan. In five consecutive patients, this pattern was able to predict primary γ-Sarcoglycan mutations. Five different mutations were found, including a recurrent novel splice mutation, a large deletion of the entire gene and a novel missense mutation (Leu90Ser). The mutation Cys283Tyr, previously restricted to Gypsy populations was found in compound heterozygosity with del521T, common in north Africa. The variety of known and novel mutations found indicates that the immunohistochemical profile of γ-Sarcoglycan mutations is not restricted to a particular mutation or type of mutation, but rather is a general reflection of the effect of γ-Sarcoglycan mutations on the composition of the Sarcoglycan complex. Complete immunohistochemical analysis with all available Sarcoglycan antibodies, therefore, is a useful tool to guide the molecular genetic investigations that are necessary to arrive at the correct genetic diagnosis in a given case.
-
Sarcoglycanopathies in Dutch patients with autosomal recessive limb girdle muscular dystrophy.
Journal of neurology, 2000Co-Authors: H.b. Ginjaar, A.j. Van Der Kooi, H. Ceelie, A. L. J. Kneppers, M. Van Meegen, Peter G. Barth, H.f.m. Busch, John H. J. Wokke, L.v.b. Anderson, Carsten G. BönnemannAbstract:Within a group of 76 sporadic/autosomal recessive limb girdle muscular dystrophy (LGMD) patients we tried to identify those with LGMD type 2C-E. Muscle biopsy specimens of 40 index patients, who had 22 affected sibs, were analyzed immuno-histochemically for the presence of three subunits: alpha-, beta-, and gamma-Sarcoglycans. Abnormal Sarcoglycan expression was established in eight patients, with six affected sibs. In one patient gamma-Sarcoglycan was absent, and both alpha- and beta-Sarcoglycans were reduced. In the remaining seven patients gamma-Sarcoglycan was (slightly) reduced, and alpha- and beta-Sarcoglycans were absent or reduced. By DNA sequencing mutations were detected in one of the three Sarcoglycan genes in all eight cases. Three patients had mutations in the alpha-, three in the beta-, and two in the gamma-Sarcoglycan gene. The patients with Sarcoglycanopathy comprised the more severely affected cases (P=0.04). In conclusion, Sarcoglycanopathy was identified in 23 % (14/62) of the autosomal recessive LGMD patients.
-
molecular organization of Sarcoglycan complex in mouse myotubes in culture
Journal of Cell Biology, 1998Co-Authors: Yiu-mo Chan, Carsten G. Bönnemann, Hart G.w. Lidov, Louis M. KunkelAbstract:The Sarcoglycans are a complex of four transmembrane proteins (α, β, γ, and δ) which are primarily expressed in skeletal muscle and are closely associated with dystrophin and the dystroglycans in the muscle membrane. Mutations in the Sarcoglycans are responsible for four autosomal recessive forms of muscular dystrophy. The function and the organization of the Sarcoglycan complex are unknown. We have used coimmunoprecipitation and in vivo cross-linking techniques to analyze the Sarcoglycan complex in cultured mouse myotubes. We demonstrate that the interaction between β- and δ-Sarcoglycan is resistant to high concentrations of SDS and α-Sarcoglycan is less tightly associated with other members of the complex. Cross-linking experiments show that β-, γ-, and δ-Sarcoglycan are in close proximity to one another and that δ-Sarcoglycan can be cross-linked to the dystroglycan complex. In addition, three of the Sarcoglycans (β, γ, and δ) are shown to form intramolecular disulfide bonds. These studies further our knowledge of the structure of the Sarcoglycan complex. Our proposed model of their interactions helps to explain some of the emerging data on the consequences of mutations in the individual Sarcoglycans, their effect on the complex, and potentially the clinical course of muscular dystrophies.
-
Mutations That Disrupt the Carboxyl-Terminus of γ-Sarcoglycan Cause Muscular Dystrophy
Human molecular genetics, 1996Co-Authors: Elizabeth M. Mcnally, Satoru Noguchi, Eijiro Ozawa, David Duggan, Marina Fanin, Carsten G. Bönnemann, J. Rafael Gorospe, Elena Pegoraro, Hart G.w. Lidov, Richard S. FinkelAbstract:Recently, mutations in the genes encoding several of the dystrophin-associated proteins have been identified that produce phenotypes ranging from severe Duchenne-like autosomal recessive muscular dystrophy to the milder limb-girdle muscular dystrophies (LGMDs). LGMD type 2C is generally associated with a more severe clinical course and is prevalent in northern Africa. A previous study identified a single base pair deletion in the gene encoding the dystrophin-associated protein gamma-Sarcoglycan in a number of Tunisian muscular dystrophy patients. To investigate whether gamma-Sarcoglycan gene mutations cause autosomal recessive muscular dystrophy in other populations, we studied 50 muscular dystrophy patients from the United States and Italy. The muscle biopsies from these 50 patients showed no abnormality of dystrophin but did show diminished immunostaining for the dystrophin-associated protein alpha-Sarcoglycan. Four patients with a severe muscular dystrophy phenotype were identified with homozygous, frameshifting mutations in gamma-Sarcoglycan. Two of the four have microdeletions that disrupt the distal carboxyl-terminus of gamma-Sarcoglycan yet result in a complete absence of gamma-and beta-Sarcoglycan suggesting the importance of this region for stability of the Sarcoglycan complex. This region of gamma-Sarcoglycan, like beta-Sarcoglycan, has a number of cysteine residues similar to those in epidermal growth factor cysteine-rich regions.
-
β Sarcoglycan a3b mutations cause autosomal recessive muscular dystrophy with loss of the Sarcoglycan complex
Nature Genetics, 1995Co-Authors: Carsten G. Bönnemann, Mikiharu Yoshida, Satoru Noguchi, Elizabeth M. Mcnally, David Duggan, Raju Modi, Yuji Mizuno, Emanuela Gussoni, C Angelini, Eric P. HoffmanAbstract:The dystrophin associated proteins (DAPs) are good candidates for harboring primary mutations in the genetically heterogeneous autosomal recessive muscular dystrophies (ARMD). The transmembrane components of the DAPs can be separated into the dystroglycan and the Sarcoglycan complexes. Here we report the isolation of cDNAs encoding the 43 kD Sarcoglycan protein beta-Sarcoglycan (A3b) and the localization of the human gene to chromosome 4q12. We describe a young girl with ARMD with truncating mutations on both alleles. Immunostaining of her muscle biopsy shows specific loss of the components of the Sarcoglycan complex (beta-Sarcoglycan, alpha-Sarcoglycan (adhalin), and 35 kD Sarcoglycan). Thus secondary destabilization of the Sarcoglycan complex may be an important pathophysiological event in ARMD.
Louis M. Kunkel - One of the best experts on this subject based on the ideXlab platform.
-
δ-Sarcoglycan is required for early zebrafish muscle organization
Experimental cell research, 2004Co-Authors: Jeffrey R. Guyon, Vincenzo Nigro, Alycia N. Mosley, Susan J. Jun, Federica Montanaro, Leta S. Steffen, Yi Zhou, Len I. Zon, Louis M. KunkelAbstract:Mutations in Sarcoglycans (alpha-, beta-, gamma-, and delta-) have been linked with limb girdle muscular dystrophy (LGMD) types 2C-F in humans. We have cloned the zebrafish orthologue encoding delta-Sarcoglycan and mapped the gene to linkage group 21. The predicted zebrafish delta-Sarcoglycan protein is highly homologous with its human orthologue including conservation of two of the three predicted glycosylation sites. Like other members of the dystrophin-associated protein complex (DAPC), delta-Sarcoglycan localizes to the sarcolemmal membrane of the myofiber in adult zebrafish, but is more apparent at the myosepta in developing embryos. Zebrafish embryos injected with morpholinos against delta-Sarcoglycan were relatively inactive at 5 dpf, their myofibers were disorganized, and swim bladders uninflated. Immunohistochemical and immunoblotting experiments show that delta-, beta-, and gamma-Sarcoglycans were all downregulated in the morphants, whereas dystrophin expression was unaffected. Whereas humans lacking delta-Sarcoglycan primarily show adult phenotypes, our results suggest that delta-Sarcoglycan plays a role in early zebrafish muscle development.
-
Specific assembly pathway of Sarcoglycans is dependent on beta- and delta-Sarcoglycan.
Muscle & nerve, 2004Co-Authors: Weixing Shi, Louis M. Kunkel, Zaili Chen, Jodi Schottenfeld, Richard C. Stahl, Yiu-mo ChanAbstract:Mutations in Sarcoglycans (SG) have been reported to cause autosomal-recessive limb-girdle muscular dystrophy (LGMD) and dilated cardiomyopathy. In skeletal and cardiac muscle, Sarcoglycans exist as a complex of four transmembrane proteins (alpha-, beta-, gamma-, and delta-SG). In this study, the assembly of the Sarcoglycan complex was examined in a heterologous expression system. Our results demonstrated that the assembly process occurs as a discrete stepwise process. We found that beta-SG appears to play an initiating role and its association with delta-SG is essential for the proper localization of the Sarcoglycan complex to the cell membrane. The incorporation of alpha-SG into the Sarcoglycan complex occurs at the final stage by interaction with gamma-SG. These findings were supported by chemical cross-linking of endogenous Sarcoglycans in cultured myotubes. We have also provided evidence that glycosylation-defective mutations in beta-SG and a common mutation in gamma-SG (C283Y) disrupt Sarcoglycan-complex formation. Our proposed model for the assembly and structure of Sarcoglycans should generate important insight into their function in muscle as well as their role in muscular dystrophies and cardiomyopathies.
-
Calpain 3 cleaves filamin C and regulates its ability to interact with gamma- and delta-Sarcoglycans.
Muscle & nerve, 2003Co-Authors: Jeffrey R. Guyon, Louis M. Kunkel, Jacques S. Beckmann, Terri G Thompson, Elena Kudryashova, Alexandra Potts, Isin Dalkilic, Melissa A. Brosius, Melissa J. SpencerAbstract:Calpain 3 (C3) is the only muscle-specific member of the calcium-dependent protease family. Although neither its physiological function nor its in vivo substrates are known, C3 must be an important protein for normal muscle function as mutations in the C3 gene result in limb-girdle muscular dystrophy type 2A. Previous reports have shown that the ubiquitous calpains (μ and m) proteolyze filamins in nonmuscle cells. This observation suggests that the muscle-specific filamin C (FLNC) is a good candidate substrate for C3. Binding studies using recombinant proteins establish that recombinant C3 and native FLNC can interact. When these two proteins are translated in vitro and incubated together, C3 cleaves the C-terminal portion of FLNC. Cleavage is specific as C3 fails to cleave FLNC lacking its C-terminal hinge and putative dimerization domains. Cotransfection experiments in COS-7 cells confirm that C3 can cleave the C-terminus of FLNC in live cells. The C-terminus of FLNC has been shown to bind the cytoplasmic domains of both δ- and γ-Sarcoglycan. Removal of the last 127 amino acids from FLNC, a protein that mimics FLNC after C3 cleavage, abolishes this interaction with the Sarcoglycans. These studies confirm that C3 can cleave FLNC in vitro and suggest that FLNC may be an in vivo substrate for C3, functioning to regulate protein–protein interactions with the Sarcoglycans. Thus, calpain-mediated remodeling of cytoskeletal–membrane interactions, such as those that occur during myoblast fusion and muscle repair, may involve regulation of FLNC–Sarcoglycan interactions. Muscle Nerve 28: 472–483, 2003
-
filamin 2 fln2 a muscle specific Sarcoglycan interacting protein
Journal of Cell Biology, 2000Co-Authors: Terri G Thompson, Andrew A. Hack, Elizabeth M. Mcnally, Yiu-mo Chan, Hart G.w. Lidov, Melissa Brosius, Michael Rajala, Simon C Watkins, Louis M. KunkelAbstract:Mutations in genes encoding for the Sarcoglycans, a subset of proteins within the dystrophin–glycoprotein complex, produce a limb-girdle muscular dystrophy phenotype; however, the precise role of this group of proteins in the skeletal muscle is not known. To understand the role of the Sarcoglycan complex, we looked for Sarcoglycan interacting proteins with the hope of finding novel members of the dystrophin–glycoprotein complex. Using the yeast two-hybrid method, we have identified a skeletal muscle-specific form of filamin, which we term filamin 2 (FLN2), as a γ- and δ-Sarcoglycan interacting protein. In addition, we demonstrate that FLN2 protein localization in limb-girdle muscular dystrophy and Duchenne muscular dystrophy patients and mice is altered when compared with unaffected individuals. Previous studies of filamin family members have determined that these proteins are involved in actin reorganization and signal transduction cascades associated with cell migration, adhesion, differentiation, force transduction, and survival. Specifically, filamin proteins have been found essential in maintaining membrane integrity during force application. The finding that FLN2 interacts with the Sarcoglycans introduces new implications for the pathogenesis of muscular dystrophy.
-
molecular organization of Sarcoglycan complex in mouse myotubes in culture
Journal of Cell Biology, 1998Co-Authors: Yiu-mo Chan, Carsten G. Bönnemann, Hart G.w. Lidov, Louis M. KunkelAbstract:The Sarcoglycans are a complex of four transmembrane proteins (α, β, γ, and δ) which are primarily expressed in skeletal muscle and are closely associated with dystrophin and the dystroglycans in the muscle membrane. Mutations in the Sarcoglycans are responsible for four autosomal recessive forms of muscular dystrophy. The function and the organization of the Sarcoglycan complex are unknown. We have used coimmunoprecipitation and in vivo cross-linking techniques to analyze the Sarcoglycan complex in cultured mouse myotubes. We demonstrate that the interaction between β- and δ-Sarcoglycan is resistant to high concentrations of SDS and α-Sarcoglycan is less tightly associated with other members of the complex. Cross-linking experiments show that β-, γ-, and δ-Sarcoglycan are in close proximity to one another and that δ-Sarcoglycan can be cross-linked to the dystroglycan complex. In addition, three of the Sarcoglycans (β, γ, and δ) are shown to form intramolecular disulfide bonds. These studies further our knowledge of the structure of the Sarcoglycan complex. Our proposed model of their interactions helps to explain some of the emerging data on the consequences of mutations in the individual Sarcoglycans, their effect on the complex, and potentially the clinical course of muscular dystrophies.
Dorianna Sandonà - One of the best experts on this subject based on the ideXlab platform.
-
Combined Use of CFTR Correctors in LGMD2D Myotubes Improves Sarcoglycan Complex Recovery
International journal of molecular sciences, 2020Co-Authors: Marcello Carotti, Martina Scano, Irene Fancello, Isabelle Richard, Giovanni Risato, Mona Bensalah, Michela Soardi, Dorianna SandonàAbstract:Sarcoglycanopathies are rare limb girdle muscular dystrophies, still incurable, even though symptomatic treatments may slow down the disease progression. Most of the disease-causing defects are missense mutations leading to a folding defective protein, promptly removed by the cell’s quality control, even if possibly functional. Recently, we repurposed small molecules screened for cystic fibrosis as potential therapeutics in Sarcoglycanopathy. Indeed, cystic fibrosis transmembrane regulator (CFTR) correctors successfully recovered the defective Sarcoglycan-complex in vitro. Our aim was to test the combined administration of some CFTR correctors with C17, the most effective on Sarcoglycans identified so far, and evaluate the stability of the rescued Sarcoglycan-complex. We treated differentiated myogenic cells from both Sarcoglycanopathy and healthy donors, evaluating the global rescue and the sarcolemma localization of the mutated protein, by biotinylation assays and western blot analyses. We observed the additive/synergistic action of some compounds, gathering the first ideas on possible mechanism/s of action. Our data also suggest that a defective α-Sarcoglycan is competent for assembly into the complex that, if helped in cell traffic, can successfully reach the sarcolemma. In conclusion, our results strengthen the idea that CFTR correctors, acting probably as proteostasis modulators, have the potential to progress as therapeutics for Sarcoglycanopathies caused by missense mutations.
-
unveiling the degradative route of the v247m α Sarcoglycan mutant responsible for lgmd 2d
Human Molecular Genetics, 2014Co-Authors: Elisa Bianchini, Marina Fanin, Romeo Betto, Kamel Mamchaoui, Dorianna SandonàAbstract:Many membrane and secretory proteins that fail to pass quality control in the endoplasmic reticulum (ER) are dislocated into the cytosol and degraded by the proteasome. In applying rigid rules, however, quality control sometimes discharges proteins that, even though defective, retain their function. The unnecessary removal of such proteins represents the pathogenetic hallmark of diverse genetic diseases, in the case of ΔF508 mutant of cystic fibrosis transmembrane conductance regulator being probably the best known example. Recently, the inappropriate proteasomal degradation of skeletal muscle Sarcoglycans (α, β, γ and δ) with missense mutation has been proposed to be at the bases of mild-to-severe forms of limb girdle muscular dystrophy (LGMD) known as type 2D, 2E, 2C and 2F, respectively. The quality control pathway responsible for Sarcoglycan mutant disposal, however, is so far unexplored. Here we reveal key components of the degradative route of V247M α-Sarcoglycan mutant, the second most frequently reported mutation in LGMD-2D. The disclosure of the pathway, which is led by the E3 ligases HRD1 and RFP2, permits to identify new potential druggable targets of a disease for which no effective therapy is at present available. Notably, we show that the pharmacological inhibition of HRD1 activity rescues the expression of V247-α-Sarcoglycan both in a heterologous cell model and in myotubes derived from a LGMD-2D patient carrying the L31P/V247M mutations. This represents the first evidence that the activity of E3 ligases, the enzymes in charge of mutant fate, can be eligible for drug interventions to treat Sarcoglycanopathy.
-
Sarcoglycanopathies: molecular pathogenesis and therapeutic prospects
Expert Reviews in Molecular Medicine, 2009Co-Authors: Dorianna Sandonà, Romeo BettoAbstract:Sarcoglycanopathies are a group of autosomal recessive muscle-wasting disorders caused by genetic defects in one of four cell membrane glycoproteins, alpha-, beta-, gamma- or delta-Sarcoglycan. These four Sarcoglycans form a subcomplex that is closely linked to the major dystrophin-associated protein complex, which is essential for membrane integrity during muscle contraction and provides a scaffold for important signalling molecules. Proper assembly, trafficking and targeting of the Sarcoglycan complex is of vital importance, and mutations that severely perturb tetramer formation and localisation result in Sarcoglycanopathy. Gene defects in one Sarcoglycan cause the absence or reduced concentration of the other subunits. Most genetic defects generate mutated proteins that are degraded through the cell's quality control system; however, in many cases, conformational modifications do not affect the function of the protein, yet it is recognised as misfolded and prematurely degraded. Recent evidence shows that misfolded Sarcoglycans could be rescued to the cell membrane by assisting their maturation along the ER secretory pathway. This review summarises the etiopathogenesis of Sarcoglycanopathies and highlights the quality control machinery as a potential pharmacological target for therapy of these genetic disorders.
-
Inhibition of Proteasome Activity Promotes the Correct Localization of Disease-Causing α-Sarcoglycan Mutants in HEK-293 Cells Constitutively Expressing β-, γ-, and δ-Sarcoglycan
The American journal of pathology, 2008Co-Authors: Stefano Gastaldello, Romeo Betto, Marina Fanin, Corrado Angelini, Simona D'angelo, Susanna Franzoso, Dorianna SandonàAbstract:Sarcoglycanopathies are progressive muscle-wasting disorders caused by genetic defects of four proteins, α-, β-, γ-, and δ-Sarcoglycan, which are elements of a key transmembrane complex of striated muscle. The proper assembly of the Sarcoglycan complex represents a critical issue of Sarcoglycanopathies, as several mutations severely perturb tetramer formation. Misfolded proteins are generally degraded through the cell's quality-control system; however, this can also lead to the removal of some functional polypeptides. To explore whether it is possible to rescue Sarcoglycan mutants by preventing their degradation, we generated a heterologous cell system, based on human embryonic kidney (HEK) 293 cells, constitutively expressing three (β, γ, and δ) of the four Sarcoglycans. In these βγδ-HEK cells, the lack of α-Sarcoglycan prevented complex formation and cell surface localization, wheras the presence of α-Sarcoglycan allowed maturation and targeting of the tetramer. As in muscles of Sarcoglycanopathy patients, transfection of βγδ-HEK cells with disease-causing α-Sarcoglycan mutants led to dramatic reduction of the mutated proteins and the absence of the complex from the cell surface. Proteasomal inhibition reduced the degradation of mutants and facilitated the assembly and targeting of the Sarcoglycan complex to the plasma membrane. These data provide important insights for the potential development of pharmacological therapies for Sarcoglycanopathies.
Daniel E Michele - One of the best experts on this subject based on the ideXlab platform.
-
dilated cardiomyopathy mutations in δ Sarcoglycan exert a dominant negative effect on cardiac myocyte mechanical stability
American Journal of Physiology-heart and Circulatory Physiology, 2016Co-Authors: Matthew D Campbell, Marc Witcher, Anoop Gopal, Daniel E MicheleAbstract:Delta-Sarcoglycan is a component of the Sarcoglycan subcomplex within the dystrophin-glycoprotein complex located at the plasma membrane of muscle cells. While recessive mutations in δ-Sarcoglycan cause limb girdle muscular dystrophy 2F, dominant mutations in δ-Sarcoglycan have been linked to inherited dilated cardiomyopathy (DCM). The purpose of this study was to investigate functional cellular defects present in adult cardiac myocytes expressing mutant δ-Sarcoglycans harboring the dominant inherited DCM mutations R71T or R97Q. This study demonstrates that DCM mutant δ-Sarcoglycans can be stably expressed in adult rat cardiac myocytes and traffic similarly to wild-type δ-Sarcoglycan to the plasma membrane, without perturbing assembly of the dystrophin-glycoprotein complex. However, expression of DCM mutant δ-Sarcoglycan in adult rat cardiac myocytes is sufficient to alter cardiac myocyte plasma membrane stability in the presence of mechanical strain. Upon cyclical cell stretching, cardiac myocytes expressing mutant δ-Sarcoglycan R97Q or R71T have increased cell-impermeant dye uptake and undergo contractures at greater frequencies than myocytes expressing normal δ-Sarcoglycan. Additionally, the R71T mutation creates an ectopic N-linked glycosylation site that results in aberrant glycosylation of the extracellular domain of δ-Sarcoglycan. Therefore, appropriate glycosylation of δ-Sarcoglycan may also be necessary for proper δ-Sarcoglycan function and overall dystrophin-glycoprotein complex function. These studies demonstrate that DCM mutations in δ-Sarcoglycan can exert a dominant negative effect on dystrophin-glycoprotein complex function leading to myocardial mechanical instability that may underlie the pathogenesis of δ-Sarcoglycan-associated DCM.
-
gene transfer establishes primacy of striated vs smooth muscle Sarcoglycan complex in limb girdle muscular dystrophy
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Shanna M Sawatzki, Rita Barresi, Valérie Allamand, Kathleen M Schmainda, Daniel E MicheleAbstract:Limb-girdle muscular dystrophy types 2E and F are characterized by skeletal muscle weakness and often cardiomyopathy and are due to mutations in the genes encoding β- and δ-Sarcoglycan. We previously demonstrated that loss of Sarcoglycans in smooth muscle leads to constrictions of the microvasculature that contributes to the cardiac phenotype. It is unclear how vasculature abnormalities affect skeletal muscle. We injected recombinant β- or δ-Sarcoglycan adenoviruses into skeletal muscles of corresponding null mice. We hypothesized that the adenoviruses would not transduce vascular smooth muscle, and we would only target skeletal muscle. Indeed, sustained expression of intact Sarcoglycan–sarcospan complex was noted at the sarcolemma, neuromuscular junction, myotendinous junction, and in peripheral nerve, but not in vascular smooth muscle. Gene transfer of the corresponding deleted Sarcoglycan gene preserved sarcolemmal integrity, prevented pathological dystrophy and hypertrophy, and protected against exercised-induced damage. We conclude that vascular dysfunction is not a primary cause of β- and δ-Sarcoglycan-deficient muscular dystrophy. In addition, we show successful functional rescue of entire muscles after adenovirus-mediated gene delivery. Thus, virus-mediated gene transfer of Sarcoglycans to skeletal muscle in combination with pharmacological prevention of cardiomyopathy constitute promising therapeutic strategies for limb-girdle muscular dystrophies.
-
Gene transfer establishes primacy of striated vs. smooth muscle Sarcoglycan complex in limb-girdle muscular dystrophy.
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Shanna M Sawatzki, Rita Barresi, Valérie Allamand, Kathleen M Schmainda, Daniel E MicheleAbstract:Limb-girdle muscular dystrophy types 2E and F are characterized by skeletal muscle weakness and often cardiomyopathy and are due to mutations in the genes encoding β- and δ-Sarcoglycan. We previously demonstrated that loss of Sarcoglycans in smooth muscle leads to constrictions of the microvasculature that contributes to the cardiac phenotype. It is unclear how vasculature abnormalities affect skeletal muscle. We injected recombinant β- or δ-Sarcoglycan adenoviruses into skeletal muscles of corresponding null mice. We hypothesized that the adenoviruses would not transduce vascular smooth muscle, and we would only target skeletal muscle. Indeed, sustained expression of intact Sarcoglycan–sarcospan complex was noted at the sarcolemma, neuromuscular junction, myotendinous junction, and in peripheral nerve, but not in vascular smooth muscle. Gene transfer of the corresponding deleted Sarcoglycan gene preserved sarcolemmal integrity, prevented pathological dystrophy and hypertrophy, and protected against exercised-induced damage. We conclude that vascular dysfunction is not a primary cause of β- and δ-Sarcoglycan-deficient muscular dystrophy. In addition, we show successful functional rescue of entire muscles after adenovirus-mediated gene delivery. Thus, virus-mediated gene transfer of Sarcoglycans to skeletal muscle in combination with pharmacological prevention of cardiomyopathy constitute promising therapeutic strategies for limb-girdle muscular dystrophies.
