expert reviews http://www.expertreviews.org/ in molecular medicine Sarcoglycanopathies: molecular pathogenesis and therapeutic prospects

Dorianna Sandona` 1 and Romeo Betto2,* Sarcoglycanopathies are a group of autosomal recessive muscle-wasting disorders caused by genetic defects in one of four cell membrane glycoproteins, a-, b-, g-ord-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.

Sarcoglycanopathies are autosomal recessive muscle. The sarcoglycan complex, whose role is muscle-wasting disorders that result only now being revealed, is a component of a from genetic defects of four transmembrane major complex formed by dystrophin at glycoproteins, a-, b-, g- and d-sarcoglycan. costameres in the cell membrane (Refs 1, 2, 3, These four subunits form a distinct complex at 4). Sarcoglycanopathies are included in a large the cell membrane of skeletal and cardiac group of limb-girdle muscular dystrophy

1Department of Biomedical Sciences, University of Padova, 35121 Padova, Italy. 2C.N.R. Institute of Neuroscience, Neuromuscular Biology and Physiopathology, 35121 Padova, Italy. *Corresponding author: Romeo Betto, C.N.R. Institute of Neuroscience, Neuromuscular Biology

and Physiopathology, Viale Giuseppe Colombo 3, 35121 Padova, Italy. Tel: +39 049 8276027; Sarcoglycanopathies: molecular pathogenesis and therapeutic prospects Fax: +39 049 8276040; E-mail: [email protected]

1 Accession information: doi:10.1017/S1462399409001203; Vol. 11; e28; September 2009 & Cambridge University Press 2009. Re-use permitted under a Creative Commons Licence–by-nc-sa. expert reviews http://www.expertreviews.org/ in molecular medicine

(LGMD) because these progressive muscle maturation process along the ER secretory disorders predominantly affect proximal pathway (Refs 13, 14). The purpose of this muscles around the scapular and the pelvic review is to provide a comprehensive overview girdles. Mutations in individual sarcoglycans of the molecular organisation of the sarcoglycan are responsible for LGMD-2C (g-sarcoglycan), complex and of the pathogenetic events causing LGMD-2D (a-sarcoglycan), LGMD-2E (b- the disease. Novel therapeutic strategies for the sarcoglycan) and LGMD-2F (d-sarcoglycan) treatment of sarcoglycanopathies based on (Refs 5, 6, 7, 8, 9). The clinical phenotype of interference of the ER-processing machinery are sarcoglycanopathies is very heterogeneous, and also evaluated. age of onset, rate of progression and severity can vary between and within affected families The dystrophin–glycoprotein complex (Ref. 10). In general, the disease is characterised The four sarcoglycans form a tetrameric by progressive weakness and degeneration of subcomplex within the multimeric complex skeletal muscle, leading to loss of ambulation, based on dystrophin in the cell membrane of difficulties in breathing and often premature skeletal and cardiac muscle. Dystrophin is a death. The majority of sarcoglycanopathies are large actin-binding cytoskeletal protein that is associated with missense mutations that essential for the organisation of the cell- generate substitution of single residues that membrane-associated dystrophin–glycoprotein could lead to a misfolded protein. Analysis of complex (DGC). The DGC is enriched in muscle samples from patients showed that costameres, which are specialised cytoskeletal these mutations result in either the complete structures connecting the plasma membrane absence or the presence of only trace amounts and Z discs of peripheral myofibrils (Refs 2, 15). of the protein in the cell membrane. Dystrophin is composed of four distinct Sarcoglycans are transmembrane proteins that functional domains: the N-terminal actin- mature in the endoplasmic reticulum (ER) binding domain, a long central domain where nascent proteins reach their native containing 24 spectrin-like repeats, a cysteine- conformation through the activity of an efficient rich domain and the C-terminal domain. The quality control system. Misfolded proteins are protein forms a tight link with the actin identified by the quality control system and cytoskeleton through the N-terminal domain, retrotranslocated to the cytosol for proteasomal an association reinforced by additional binding degradation through the ER-associated protein sites within some spectrin repeats (Ref. 1). At degradation (ERAD) pathway (Refs 11, 12). the distal region of the protein, dystrophin Occasionally, defective proteins may pass forms, via its cysteine-rich domain, a tight the quality control and reach the cell association with the transmembrane protein membrane, where, since they are nonfunctional b-dystroglycan, which in turn is strongly linked and thus unstable, they are dismantled and to the extracellular a-dystroglycan (Fig. 1). The degraded. Defects in each sarcoglycan have two dystroglycans are products of a single gene destabilising consequences on the entire that is post-translationally cleaved into two sarcoglycan complex. Therefore, the subunits (Ref. 17). The highly glycosylated a- pathogenetic mechanisms may comprise: (1) the dystroglycan forms a strong interaction with processing of defective sarcoglycan subunits; (2) laminin a2, a major constituent of muscle fibre the inability to assemble into a complete basement membrane. Thus, the backbone complex; or (3) the targeting of a dysfunctional formed by dystrophin, dystroglycans and sarcoglycan complex to the cell membrane. laminin a2 represents a transmembrane Sequence analysis of sarcoglycans indicated structure directly connecting the actin that although many missense mutations might cytoskeleton to the extracellular matrix. The not have functional consequences, they are localisation of DGC in line with costameres is intercepted by the quality control system, assured by the association of b-dystroglycan which significantly slows their processing and and dystrophin to two ankyrins (B and G), results in the disposal of the mutant protein. which are components of the subsarcolemmal

Recent evidence shows that these misfolded cytoskeleton (Ref. 18). The DGC is thus thought Sarcoglycanopathies: molecular pathogenesis and therapeutic prospects ‘functional’ sarcoglycans could be rescued to to provide structural support to the plasma the cell membrane by assisting them in the membrane and to protect it from the 2 Accession information: doi:10.1017/S1462399409001203; Vol. 11; e28; September 2009 & Cambridge University Press 2009. Re-use permitted under a Creative Commons Licence–by-nc-sa. expert reviews http://www.expertreviews.org/ in molecular medicine

Laminin α2

α-Dystroglycan

Putative partners of DGC Sarcoglycans α O-glycan Extracellular β Biglycan δ γ Perlecan N-glycan Rapsyn β Transmembrane -Dystroglycan Integrins Plasma membrane 16 kDa vH+-ATPase Caveolin Intracellular Sarcospan γ-filamin Syncoilin nNOS Ankyrin Spectrin Dystrophin Syn Plectin Dystrobrevin Syn

Actin filament

The dystrophin–glycoprotein complex Expert Reviews in Molecular Medicine © Cambridge University Press 2009

Figure 1. The dystrophin–glycoprotein complex. Simplified scheme of dystrophin–glycoprotein complex (DGC) organisation. Dystrophin is localised in the cytoplasmic face of skeletal and cardiac cell membranes. Dystrophin binds actin filaments through two specific binding sites in its C-terminal domain and sites in the spectrin-like-repeat portion. The cysteine-rich domain assures the binding of dystrophin to the transmembrane b-dystroglycan, which in turn associates extracellularly with a-dystroglycan (blue box). a- dystroglycan interacts with laminin a2 and other cell matrix components, completing the backbone of the DGC. The sarcoglycan–sarcospan complex (light blue box), forms a lateral association with dystroglycans. N-glycan and O-glycan indicate, respectively, the N- and O-glycosylation moieties post-translationally added to sarcoglycans and dystroglycans. Additional sarcoglycan partners have been proposed (not all indicated), both intracellularly and extracellularly. Dystrobrevin, syntrophin (Syn) and neuronal nitric oxide synthase (nNOS) are intracellular components of the DGC. Many other proteins have been indicated to interact with DGC, either permanently or dynamically; a few of these are indicated. For a complete list of DGC components the reader should refer Refs 1, 2, 15, 16. mechanical stress of contractile activity by (DMD) and the milder Becker muscular transmitting the lateral tension generated by dystrophy (BMD), whereas defects of laminin muscle contraction to the extracellular matrix a2 and a-dystroglycan glycosylating enzymes (Refs 15, 19). The critical protective role of cause diverse forms of muscular dystrophy dystrophin and of the entire membrane- (Refs 2, 20, 21). With a defective DGC, the associated DGC is demonstrated by the fact that backbone structure is dismantled so that the

genetic defects of dystrophin are responsible for cell membrane becomes exposed to muscle Sarcoglycanopathies: molecular pathogenesis and therapeutic prospects the most common type of muscular dystrophy, contraction stresses. As a consequence, cell the incurable Duchenne muscular dystrophy membrane focal ruptures might occur, leading 3 Accession information: doi:10.1017/S1462399409001203; Vol. 11; e28; September 2009 & Cambridge University Press 2009. Re-use permitted under a Creative Commons Licence–by-nc-sa. expert reviews http://www.expertreviews.org/ in molecular medicine to transient intracellular calcium influx, which biglycan and g-filamin, however, is not well triggers a series of pathogenic events that result understood. Recently, aquaporin 4, a muscle- in muscle degeneration and the dystrophic specific water channel, was shown to be phenotype (Refs 2, 21). associated with sarcoglycan through a1- syntrophin, an interaction validated by its Role of sarcoglycans in the dystrophin– severe reduction in muscles of glycoprotein complex sarcoglycanopathy patients (Ref. 32). Loss of sarcoglycan is the specific cause of Altogether, these data demonstrate that the sarcoglycanopathy, whereas the absence of sarcoglycan complex forms multiple interactions dystrophin in DMD causes instability of the with the other DGC components and enforce the complex and also leads to the severe reduction view of its crucial role in stabilising the whole of sarcoglycans. Thus, an altered sarcoglycan DGC structure. However, the sarcoglycans might complex is a common trait of the two muscular also be involved in signal transduction (Refs 2, 3, dystrophies and probably represents an 21, 33), a role that is primarily supported by the aggravating condition in DMD. anchorage provided by the complex to nNOS The role of the four sarcoglycans in the through dystrobrevin and syntrophin (Ref. 25). molecular organisation of the DGC is not yet Moreover, the sarcoglycan complex has been well defined. The sarcoglycan complex is shown to cooperate with integrins in mediating known to form a tight side-association with cell adhesion to the extracellular matrix (Ref. 34). dystroglycan (Refs 22, 23), but is also involved In support of this, integrins are upregulated in composite molecular relationships with other when the complex is missing (Ref. 35). The constitutive DGC elements such as a- sarcoglycan complex also interacts with the 16 dystrobrevin and syntrophin (Ref. 24), neuronal kDa subunit of vacuolar Hþ-ATPase, and thence nitric oxide synthase (nNOS) (Ref. 25) and with b1-integrin (Ref. 36) – a finding that sarcospan (Ref. 26) (Fig. 1). Sarcospan is an further supports bidirectional signalling with integral component of DGC, which directly integrins (Ref. 34). Notably, it has been interacts with and stabilises sarcoglycans. The demonstrated that the cytoplasmic tail of g- relevance of this transmembrane protein is, sarcoglycan is phosphorylated after mechanical however, still uncertain, because ablation of the stimulation, suggesting that it is a signalling sarcospan gene in mice does not result in sensor of contractility (Ref. 37). In addition, a- myopathy (Ref. 27). The sarcoglycan complex, sarcoglycan possesses an ATP-binding site, excluding g-sarcoglycan, is thought to form an conferring ATP-hydrolysing activity to the association with a-dystroglycan (Ref. 23) via an protein (Refs 38, 39). This activity suggests a extracellular proteoglycan, biglycan (Ref. 28). possible role in modulating the signalling The intracellular tail of b- and d-sarcoglycan initiated by extracellular nucleotides and a role seems to associate directly with the C-terminus in the extracellular ATP-dependent modulation of dystrophin (Ref. 29), whereas the N-terminal of skeletal muscle contractility (Ref. 40). The region of a-dystrobrevin secures sarcoglycans critical role of the sarcoglycan complex, whether to dystrophin (Ref. 24). a-Dystrobrevins, mechanical or signalling, is further corroborated syntrophin and nNOS form a signalling subunit by a recent study showing that the complete associated with dystrophin through the C- absence of the sarcoglycan complex in mice terminal domain of dystrobrevin. Additional lacking both d-sarcoglycan and dystrophin proteins seem to interact with sarcoglycans at exacerbates the pathological course (Ref. 41). This the cell membrane. Two hybrid screens evidence also suggests that the residual identified g-filamin as a g- and d-sarcoglycan- expression of the sarcoglycan complex contributes interacting protein (Ref. 30) and, as a result, g- to the milder phenotype of dystrophin-deficient filamin is reduced in LGMD-2C (g-sarcoglycan) mdx mice compared with that of patients with but not in LGMD-2D (a-sarcoglycan) (Ref. 31). DMD (Ref. 41). Since g-filamin is an actin-binding protein, it provides, through the interaction with the The sarcoglycan complex in

sarcoglycan complex, additional structural sarcoglycanopathies Sarcoglycanopathies: molecular pathogenesis and therapeutic prospects linkages between the DGC and the actin Studies of patient muscle biopsies provide cytoskeleton. The precise physiological role of additional important information on the 4 Accession information: doi:10.1017/S1462399409001203; Vol. 11; e28; September 2009 & Cambridge University Press 2009. Re-use permitted under a Creative Commons Licence–by-nc-sa. expert reviews http://www.expertreviews.org/ in molecular medicine sarcoglycan complex and its contribution to the localisation of dystrophin, dystroglycan and molecular organisation of the DGC. Analyses of laminin-a2 are unaffected (Refs 62, 65, 66, 67). muscle biopsies from sarcoglycanopathy d-Sarcoglycan-deficient animals develop patients demonstrated that the absence or cardiomyopathy, which is rarely observed in reduced expression of one sarcoglycan has LGMD-2F patients, with loss of the sarcoglycan important, but variable consequences for the complex even in smooth muscle (Ref. 60); b- stability of the other remaining components at and g-sarcoglycan-deficient mice, but not a- the cell membrane. In general, the most sarcoglycan-null mice, also display some clinically severe course was observed in cardiac involvement. patients in which the involved sarcoglycan was The sarcoglycan family of proteins in flies is absent, with a variable milder phenotype when simpler than in vertebrates: the fly genome residual protein is present. Early studies on encodes a single orthologue of vertebrate a- patients affected by sarcoglycanopathies and 1-sarcoglycans (a1-sarcoglycan), a b- reported that mutations in one sarcoglycan sarcoglycan and a single orthologue of g- and gene led to the reduction or absence of the d-sarcoglycans (gd-sarcoglycan). In Drosophila,a other sarcoglycans, with negligible tetrameric organisation of the complex is consequences for the DGC (Refs 6, 7, 8, 42, 43, suggested, with the association of a1- 44, 45, 46, 47, 48, 49). By contrast, more recent sarcoglycan, b-sarcoglycan and two copies of evidence shows that the absence of individual gd-sarcoglycan (Ref. 68). A gd-sarcoglycan- sarcoglycans has indirect consequences on DGC deficient model has been generated, which stability. In LGMD-2E and -2F patients, defects caused skeletal and cardiac defects (Ref. 69); in genes encoding b- and d-sarcoglycan results however, the fate of the other sarcoglycans was in the absence of the four sarcoglycans from the not determined. Orthologues of sarcoglycan plasma membrane and the reduction of genes have also been identified in zebrafish, dystrophin and dystroglycan (Refs 50, 51, 52, which represent an established model of 53, 54). In LGMD-2D patients, defects in a- vertebrate development (Ref. 70). Knockdown sarcoglycan causes the secondary loss of b- and of d-sarcoglycan caused skeletal and cardiac d-sarcoglycan and a severe reduction in g- development defects and reduced the levels of sarcoglycan, which can be absent altogether in b- and g-sarcoglycan (Ref. 71). more severely affected patients; b-dystroglycan In summary, muscle analysis of various LGMD and dystrophin were reduced in most cases patients and of related animal models shows that (Refs 50, 52, 53, 54). In g-sarcoglycan-deficient the absence of or defects in b- and d-sarcoglycan patients (LGMD-2C) trace amounts of residual has dramatic consequences on the expression sarcoglycans are evident at the cell membrane, of all other subunits, whereas defects in whereas no consequences for the DGC or a g-sarcoglycan have the least effect on reduction of dystrophin are reported (Refs 50, expression of the other sarcoglycans. 52, 53, 54, 55, 56). The absence of the Interestingly, the absence of a-sarcoglycan in sarcoglycan complex in muscle of patients humans causes the absence or severe reduction affected by these diseases is reported to expose of g-sarcoglycan, but b- and d-sarcoglycan are b-dystroglycan to the activity of a matrix slightly spared. By contrast, in a-sarcoglycan- metalloproteinase, thus providing an deficient mice, the other sarcoglycans explanation for its reduced level in are completely lost. Interestingly, expression sarcoglycanopathies (Ref. 57). of g-sarcoglycan and, to a lesser extent, Animal models exist for each of the four a-sarcoglycan, is reduced more than b- and sarcoglycanopathies and show similar results d-sarcoglycan across all sarcoglycanopathies. to the human situation. The absence of a-, b- and d-sarcoglycan causes the complete loss of The molecular organisation of the the other three sarcoglycans and some sarcoglycan complex destabilisation of dystroglycans (Refs 23, 58, 59, Data from LGMD patients and related animal 60, 61, 62, 63, 64). By contrast, the absence of g- models demonstrate that the four sarcoglycans

sarcoglycan in mice weakens the stability of b- have an important role at the plasma Sarcoglycanopathies: molecular pathogenesis and therapeutic prospects and d-sarcoglycan and causes severe reduction membrane, but only when they exist as a of a-sarcoglycan, but the expression and complex. A better understanding of sarcoglycan 5 Accession information: doi:10.1017/S1462399409001203; Vol. 11; e28; September 2009 & Cambridge University Press 2009. Re-use permitted under a Creative Commons Licence–by-nc-sa. expert reviews http://www.expertreviews.org/ in molecular medicine complex organisation, processing and function is four cysteine residues and one putative N- thus central to improve our knowledge of the linked glycosylation site in its extracellular pathogenic mechanisms responsible for the portion, whereas d-sarcoglycan has four dystrophic phenotype. cysteine residues and three putative N-linked Sarcoglycans are single-pass transmembrane glycosylation sites. The C-terminus of b-, d- and proteins, with a short intracellular tail and g-sarcoglycan has three conserved cysteine a large extracellular glycosylated portion that residues in a fixed position that is similar to is rich in conserved cysteine residues. those in epidermal growth factor (EGF), Six sarcoglycans have been cloned so far: a-, b-, suggesting a receptor-like function for an g-, d-, 1- and z-sarcoglycan (Table 1). a- and unknown agonist (Refs 3, 29). It is worth noting g-sarcoglycan are expressed exclusively in that the presence of a putative N-linked skeletal and cardiac muscle, whereas 1-, b-, d- glycosylation site in a protein does not and z-sarcoglycan are more widely distributed necessarily equate to a real glycosylation event. (Refs 5, 6, 7, 8, 9, 72, 73, 74). The a- and 1- In fact, although the molecular masses of sarcoglycans, are type I membrane proteins unglycosylated g- and d-sarcoglycan are with an extracellular N-terminal domain, identical (32 kDa), the presence of three N- whereas b-, g-, d- and z-sarcoglycans are type II glycans in d-sarcoglycan is expected to generate membrane proteins, with an extracellular C- a larger mass than that of g-sarcoglycan, which terminal domain. a- and 1-sarcoglycan are has one N-linked glycosylation site. Therefore, highly homologous, as are b-, g-, d- and z- either only one of the d-sarcoglycan N-linked sarcoglycan, with the latter being functionally glycosylation sites is eventually glycosylated or more similar to g-sarcoglycan than to d- the two proteins are modified differently upon sarcoglycan (Ref. 75). A cysteine cluster is post-translation processing. present in the extracellular domain of all It is widely assumed that sarcoglycans are sarcoglycans and is predicted to be important organised at the cell membrane as a tetramer, in for the tertiary structure of the protein and a complex that is exclusively expressed in for assembly of the whole complex (Ref. 22). striated muscles; this is consistent with the All four sarcoglycans possess putative notion that mutations in a-, b-, g- and d- phosphorylation sites in the intracellular sarcoglycan are responsible for muscle domain, indicating a possible post-translational sarcoglycanopathies. Mutations in the gene modulation of the protein or complex. encoding 1-sarcoglycan are associated with Protein sequence analysis shows that a- myoclonus dystonia syndrome (Ref. 77), sarcoglycan contains an N-terminal signal whereas no disease has been so far associated sequence, one transmembrane domain, five with mutations in the z-sarcoglycan gene. extracellular cysteines and two putative N- Organisation of the sarcoglycan complex occurs linked glycosylation sites, which are conserved in a strict equimolar stoichiometry (Ref. 47), in all species (Table 1 and Fig. 5). Cleavage of although atypical organisation and localisation the signal sequence and glycosylation generate of sarcoglycans have also been reported. For a protein of about 50 kDa. In the N-terminal example, in human smooth muscle, a portion, a-sarcoglycan has a cadherin-like pentameric or exameric sarcoglycan complex domain and Ca2þ-binding pockets, which have has been proposed (Ref. 78). Moreover, d- and also been described in a-dystroglycan, g-sarcoglycan seem to have an additional suggesting a possible Ca2þ-dependent intracellular membrane localisation, but any heterotypic adhesion between the two proteins contribution to muscle physiology and/or (Ref. 76). In addition, a-sarcoglycan has a pathology is unknown (Ref. 79). putative ATP-binding sequence in the extracellular domain, which is conserved in all Assembly and trafficking of the species (Ref. 38). The predicted molecular mass sarcoglycan complex of glycosylated b-sarcoglycan is about 43 kDa Studies on the assembly of the sarcoglycan and its large extracellular portion has five complex during the early stage of myotube

cysteine residues and three putative N-linked differentiation show that sarcoglycans are Sarcoglycanopathies: molecular pathogenesis and therapeutic prospects glycosylation sites. The mass of glycosylated g- cotranslationally translocated in the ER, where and d-sarcoglycan is ~35 kDa. g-sarcoglycan has they associate during transport through the 6 Accession information: doi:10.1017/S1462399409001203; Vol. 11; e28; September 2009 & Cambridge University Press 2009. Re-use permitted under a Creative Commons Licence–by-nc-sa. expert reviews http://www.expertreviews.org/ in molecular medicine

Table 1. Human sarcoglycan protein data

Protein Gene Chromosome Disease AA Massa NC a-Sarcoglycan SGCA 17q21.33 LGMD-2D 387 50 2 5

b-Sarcoglycan SGCB 4q11 LGMD-2E 318 43 3 5 g-Sarcoglycan SGCG 13q12 LGMD-2C 291 35 1 4

d-Sarcoglycan SGCD 5q33.3 LGMD-2F 290 35 3 4 1-Sarcoglycan SGCE 7q21.3 MDS 405 45 1 4

z-Sarcoglycan SGCZ 8p22 Unknown 299 35 1 4 aObserved molecular mass (kDa) based on results from SDS-PAGE. Abbreviations: LGMD, limb-girdle muscular dystrophy; MDS, Myoclonus–dystonia syndrome; AA, number of amino acid residues; N, number of putative N-linked glycosylation sites in the extracellular portion of the protein, according to http://www.uniprot.org; C, number of cysteine residues.

Golgi to the plasma membrane (Refs 22, 80) where budding of vesicles coated with coat (Fig. 2). protein complex II takes place and by which In the ER, the quality control machinery they enter the Golgi complex (Fig. 3d). facilitates polypeptide folding to ensure that Trafficking of transmembrane multimeric nascent proteins assume the correct complexes requires more elaborate processing conformation, and, if necessary, it recycles the because the individual membrane proteins proteins until the native conformation is are usually assembled in the ER and then obtained (Fig. 3a–c). Glycosylation of proteins transported as a unit to the Golgi and the cell such as sarcoglycans is performed by an membrane (Fig. 2). Evidence indicates that oligosaccharyltransferase, which attaches a multimeric complexes may also encounter post- composite oligosaccharide composed of three ER quality control checkpoints that monitor and glucose, nine mannose and two n-acetyl assist the oligomerisation process, probably glucosamine residues (N-glycan) to the nascent at ER–Golgi intermediate and cis-Golgi polypeptide. Subsequently, two glucoses are compartments (Ref. 11). Experimental evidence sequentially trimmed from the N-glycan by in CHO cells demonstrated that the glucosidase I and II, while two lectin simultaneous synthesis of the four sarcoglycans chaperons, calnexin and calreticulin, associate is mandatory for proper assembly and cell with the monoglycosylated proteins to attain membrane localisation of the sarcoglycan their pre-native conformation (Refs 11, 12). complex (Ref. 82). Biochemical analyses in Protein folding is then performed by C2C12 myogenic cells showed that sarcoglycans oxidoreductases of the PDI family and associate to form a complex in the ER as peptidyl-propyl cis/trans isomerases, which soon as they are synthesised. Thereafter, facilitate the oxidative folding of the protein, the sarcoglycan complex associates with formation and isomerisation of disulfide bonds, dystroglycans and sarcospan only during the correct cis or trans isomerisation and transportation from the Golgi to the cell prevent the formation of aggregates (Ref. 12). membrane (Ref. 83). The latter finding implies At the end of the calnexin–calreticulin cycle, that sarcoglycans, sarcospan and dystroglycan the enzyme UDP-glucose glycoprotein may exit the ER independently and then glucosyltransferase (UGGT) reattaches a glucose associate during transport to the cell membrane to imperfectly folded proteins, causing their (Ref. 84) (Fig. 2). The association of the reintroduction into the calnexin and calreticulin sarcoglycan complex with dystroglycans en cycle (Ref. 81) (Fig. 3b). Properly folded route to the cell membrane probably favours

proteins are ignored by UGGT and proceed the subsequent recruitment of cytosolic Sarcoglycanopathies: molecular pathogenesis and therapeutic prospects along the maturation pathway (Fig. 3c). Export dystrophin once it reaches its destination. In of native proteins occurs at ER-specific sites primary mouse myotubes, it was demonstrated 7 Accession information: doi:10.1017/S1462399409001203; Vol. 11; e28; September 2009 & Cambridge University Press 2009. Re-use permitted under a Creative Commons Licence–by-nc-sa. expert reviews http://www.expertreviews.org/ in molecular medicine

that b-, d- and g-sarcoglycans form a strong interaction, whereas a-sarcoglycan seems to be less tightly associated (Ref. 22). The overall Sarcoglycans evidence indicates the following sequential α events for sarcoglycan association: (1) b- β sarcoglycan initiates a strong interaction with d- δ γ β N-glycan -dystroglycan sarcoglycan; (2) g-sarcoglycan is added to the bd-sarcoglycan core; (3) a-sarcoglycan is recruited by interacting with g-sarcoglycan Plasma (Refs 22, 85). An alternative association membrane Out sequence has also been proposed, where a- sarcoglycan directly interacts with the bd- In sarcoglycan core (Refs 86, 87). An intriguing Sarcospan finding in COS-1 (Ref. 85) and in HEK293 (Ref. 86) cells shows that b- and d-sarcoglycans might be transported to the cell membrane even when expressed without a- and g-sarcoglycan. In any case, these data reinforce the view of the fundamental role of the bd-sarcoglycan core in Golgi the assembly process of the complex. However, the precise molecular interactions controlling assembly of the four sarcoglycans remain undefined. A recent study in COS-1 cells identified novel functional domains in the extracellular portion of sarcoglycans that seem to regulate association and favour transport to the cell membrane. In particular, regions of the extracellular portion close to the transmembrane domain appear to be important for regulation of the interaction between b- and d-sarcoglycan and a- and g-sarcoglycan, whereas a more distal region seems to control ER the interaction between b- and g-sarcoglycan (Ref. 29). Moreover, these authors also demonstrate that N-glycosylation and the cysteine-rich domain of d-sarcoglycan are

Figure 2. Trafficking of the sarcoglycan complex. The four sarcoglycans (a, b, g and d) are synthesised in the ER where they undergo cotranslational glycosylation (N-glycan) and proper conformational folding. Association of the four sarcoglycans occurs in the ER, with the bd- sarcoglycan core being the trigger for the assembly of the tetrameric complex. The complex is then transported to the plasma membrane through the Golgi system, where it most likely Trafficking of the sarcoglycan complex assembles with sarcospan and dystroglycan. Expert Reviews in Molecular Medicine Once in the cell membrane, the other © Cambridge University Press 2009 cytoplasmic components of the dystrophin– Sarcoglycanopathies: molecular pathogenesis and therapeutic prospects glycoprotein complex (DGC), aggregate to form the final structure. 8 Accession information: doi:10.1017/S1462399409001203; Vol. 11; e28; September 2009 & Cambridge University Press 2009. Re-use permitted under a Creative Commons Licence–by-nc-sa. expert reviews http://www.expertreviews.org/ in molecular medicine

d Export

ER Proteasome

Sarcoglycan complex f RT ERAD c U U U U R Ubiquitin Native sarcoglycan

N-glycan

UGGT M Terminally Misfolded misfolded sarcoglycan e sarcoglycan

CNX

ab

Putative ER processing of wild-type and mutant sarcoglycans Expert Reviews in Molecular Medicine © Cambridge University Press 2009

Figure 3.PutativeERprocessing of wild-typeand mutant sarcoglycans. Nascentsarcoglycanselongateinto the ER where they undergo cotranslational glycosylation and proper conformational folding (a). Non-native polypeptides may experience repetitive rounds of folding within the calnexin (CNX)–calreticulin cycling system, a process regulated by UDP-glucose glycoprotein glucosyltransferase (UGGT) (b). A successfully folded protein then enters the maturation process, which comprises assembly with the other sarcoglycans to form a tetrameric complex (c). The protein complex is then exported from the ER (d). Misfolded proteins are degraded by endoplasmic reticulum-associated degradation (ERAD) by intervention of mannosidase I (M) (e). Individual components not able to assemble are also degraded (dotted arrow). ERAD occurs in a composite process of recognition (R), targeting for ER retrotranslocation (RT), and degradation of the terminally misfolded protein by the ubiquitin–proteasome system (f). crucial for the localisation of sarcoglycans to HEK293 and HeLa cells (Refs 39, 88) but not in the cell membrane, and suggest that the COS-1 cells from monkey or CHO cells from intracellular tails of b- and d-sarcoglycan hamster (Refs 82, 85). Moreover, a-sarcoglycan interact with dystrophin (Ref. 29). cell membrane localisation seems to be The individual heterologous expression of dynamic, because the protein recycles from a-sarcoglycan, but not of the other subunits, the membrane to endosomes (Ref. 88). This results in the localisation of the protein at evidence suggests that undefined sequence

the cell membrane even in the absence of motifs of human a-sarcoglycan might permit its Sarcoglycanopathies: molecular pathogenesis and therapeutic prospects the remaining sarcoglycans. Surprisingly, this preferential solitary trafficking to the cell occurs in cells of human origin, such as membrane. Conversely, it is possible that the 9 Accession information: doi:10.1017/S1462399409001203; Vol. 11; e28; September 2009 & Cambridge University Press 2009. Re-use permitted under a Creative Commons Licence–by-nc-sa. expert reviews http://www.expertreviews.org/ in molecular medicine difference might reside in the maturation, sorting loss of function or instability of the entire and trafficking rules of the human protein-export sarcoglycan complex. In this case, the whole apparatus. This possibility is validated by recent structure will be dismantled and undergo evidence showing that the R77C a-sarcoglycan lysosomal–proteasomal degradation. The mutant, the most common LGMD-2D mutation, residual expression level of the remaining wild- remains trapped in the ER when expressed in type sarcoglycans probably depends on the human cells, either alone or together with the association rules and trafficking possibilities other sarcoglycans (Refs 13, 14, 86). By contrast, described above. However, the presence of in knock-in mice homozygous for the H77C residual expression of a mutant protein in the mutation (in mice, R at position 77 is cell membrane suggests that the mutant might conservatively substituted by H), a-sarcoglycan escape the ER quality control system, at least in and the entire complex is correctly localised at part, and forms a tetramer that is transported to the cell membrane and mice did not develop the cell membrane. The resulting dystrophic muscular dystrophy (Refs 13, 89). Since no phenotype might depend on insufficient levels of information on the tertiary structure of a- either the protein or the complete complex, or to sarcoglycan is available, it is possible that the loss-of-function consequences. protein region around H77 in mice might differ According to the Leiden Open Variation from that in humans such that this substitution Database (Ref. 91) (http://www.lovd.org), and could be considered a polymorphism in mice. to recent evidence (Refs 53, 92), at least 176 Intriguingly, when human R77C a-sarcoglycan sequence variants in the coding region of is transfected into muscles of a-sarcoglycan- sarcoglycans have been so far reported to knockout mice some aggregates do form, but cause LGMD-2C to LGMD-2F. Seventy-one the majority reaches the cell membrane are mutations that generate a truncated or (Refs 13, 89). aberrant protein or lead to nonsense-mediated decay of the transcript, whereas 105 are Pathogenetic mechanisms missense mutations that cause protein In sarcoglycanopathy, mutations in any of the misfolding. Usually, mutated misfolded four sarcoglycan genes cause a reduction or proteins synthesised in the ER are intercepted, the absence of the defective protein, and retrotranslocated in the cytosol and delivered secondarily affect expression of the other for dismantling by the ubiquitin–proteasome subunits. A reduction in protein levels can have through the multifaceted ERAD system (Fig. 4). several causes, such as defects that hamper Recognition of misfolded proteins is quite gene expression or lead to aberrant maturation rapid, and proteins are delivered for of the mRNA transcript, or defects in protein degradation in a ubiquitin-dependent manner, processing (Ref. 90), where its fate then without any detectable lag following their depends on the ER quality control system. synthesis. The process is intended to prevent Complete absence of a protein can result from the accumulation of defective proteins and nonsense mutations, which instead of formation of toxic aggregates that might block producing truncated sarcoglycans, trigger trafficking in the secretory pathway. However, nonsense-mediated mRNA decay. Absence of a several misfolded proteins are reported to be protein might also result from missense prone to aggregation, as is the case for many mutations that lead to production of misfolded myofibrillar myopathies (Ref. 93) and proteins, which are then recognised and neurodegenerative disorders, such as retained in the ER, and after unsuccessful Parkinson, Alzheimer and prion-associated cycling in the calnexin and calreticulin system, diseases (Ref. 94). are ultimately degraded through the ubiquitin– Recently, two different groups have proteasome system (Fig. 3e, f). A mutant protein demonstrated the involvement of the cell might evade the quality control mechanism, quality control system in the pathogenesis of but since it will probably not be able to form sarcoglycanopathies in heterologous cell models a stable tetramer it will still be degraded (Refs 13, 14). Both papers show that different

(Fig. 3c, f). Finally, a mutant sarcoglycan protein a-sarcoglycan mutants with single amino acid Sarcoglycanopathies: molecular pathogenesis and therapeutic prospects might fold and form a tetramer, exit the ER and substitutions are ubiquitinated and rapidly reach the cell membrane, where it could cause degraded by the proteasome. Intriguingly, 10 Accession information: doi:10.1017/S1462399409001203; Vol. 11; e28; September 2009 & Cambridge University Press 2009. Re-use permitted under a Creative Commons Licence–by-nc-sa. expert reviews http://www.expertreviews.org/ in molecular medicine

Misfolded sarcoglycan ER

N-glycan

Step 2 Step 1 M GRP94 EDEM BiP Os9

Demannosylated Step 3 N-glycan SEL1L SEC61 Cytosol

E1 HRD1 U E2 U U Derlins U U Ubiquitin U Step 4 U p97 Step 5 U Step 7 U U Step 8 Step 6

Putative progression of sarcoglycan mutants through the ER–associated degradation pathway Expert Reviews in Molecular Medicine © Cambridge University Press 2009

Figure 4. Putative progression of sarcoglycan mutants through the ER-associated degradation pathway. The putative processing of sarcoglycan mutants is represented in consecutive steps. Non-native sarcoglycans are targeted to ERAD by the intervention of mannosidase I and/or mannosidase-like proteins (EDEM), which remove a mannose from the N-glycan and terminate its maturation process (Step 1). Then, the misfolded sarcoglycan is identified and escorted for retrotranslocation by a targeting complex composed of three probable components (BiP, GRP94 and Os9) (Step 2). Dislocation of the ERAD substrate occurs through the SEL1L–HRD1 complex (Step 3), which also includes Sec61, the actual channel. During retrotranslocation, the polypeptide is ubiquitinated by cytosolic and membrane-associated E1, E2 and E3 enzymes (Step 4). Note that HRD1 is one such E3 ligase. A multimeric protein complex, which comprises the p97 ATPase and Derlin proteins (through which the complex attaches to the ER membrane), dislocates the terminally misfolded sarcoglycan from the ER (Step 5). The same p97 complex targets the polyubiquitinated (U) sarcoglycan to the 26S proteasome. Approaching the 19S cap of the proteasome, the N-glycan and the ubiquitin chain are removed by N-glycanase (Step 6) and deubiquitinating enzymes (Step 7). The polypeptide is eventually threaded into the 20S proteasome catalytic core where it is broken down into small fragments (Step 8). proteasomal inhibition by MG132 or bortezomib other sarcoglycan components, which favours (Velcade) was able to rescue expression of D97G, their transport to the cell membrane. Moreover, R98H, P228Q and V247M, but not R77C, these data suggest that D97G, R98H, P228Q and a-sarcoglycan mutants, and more importantly, V247M a-sarcoglycan mutants maintain the the mutants formed a stable sarcoglycan ability to form a potentially functional complex complex that localised at the cell membrane at the cell membrane. Regarding the inability (Ref. 14). This finding demonstrates that to recover the R77C mutation, it is worth inhibition of the proteasome, the last step in the mentioning that the defective protein is known

ERAD pathway (Fig. 4, Step 8), has a retrograde to form aggregates and is retained in the ER Sarcoglycanopathies: molecular pathogenesis and therapeutic prospects effect on mutant processing, permitting the when expressed in heterologous cells (Refs 13, assembly of partly misfolded proteins with the 14, 88); however, R77C a-sarcoglycan can 11 Accession information: doi:10.1017/S1462399409001203; Vol. 11; e28; September 2009 & Cambridge University Press 2009. Re-use permitted under a Creative Commons Licence–by-nc-sa. expert reviews http://www.expertreviews.org/ in molecular medicine occasionally be detected at the cell membrane, However, several mutants might retain their either with or without proteasome inhibitor function if they could escape the quality control treatment (Ref. 14), suggesting that the R77C system. Mutations of critical residues within mutant might escape the ERAD system. hypothetical functional domains and in highly Importantly, to prevent degradation of conserved sequences may be expected to have sarcoglycan mutants, it has been shown that it loss-of-function consequences. By contrast, is also possible to intervene upstream of the substitutions in noncritical regions might proteasome. Mannosidase I is a key ER enzyme generate functional proteins. Below, we provide a involved in retrotranslocation of terminally concise summary of mutations in the four misfolded proteins (Fig. 4, Step 1). Inhibition of sarcoglycans. a-mannosidase I, with the selective inhibitors kifunensine and deoxymannojirimycine, Mutations in a-sarcoglycan favoured the localisation of the R77C a- Seventy-three mutations have been reported in sarcoglycan mutant at the cell membrane in the a-sarcoglycan gene (SGCA) that cause HER911 cells cotransfected with b-, g-andd- changes in the protein. Fifty-two are missense sarcoglycan (Ref. 13). However, in mouse, the mutations that generate a complete protein with substitution of H77 with a cysteine does not lead a single residue substitution (Fig. 5), whereas 21 to a pathological phenotype (Refs 13, 89), mutations (nucleotide substitution, duplication, suggesting that if there are no differences in the deletion or insertions) produce truncated, protein quality control system among species, the incomplete or anomalous proteins. All but one C77varianthasnoeffectona-sarcoglycanfunction. of the a-sarcoglycan missense mutations are Recent evidence indicates that pharmacological mapped in the extracellular domain – a critical therapies could assist ER protein processing of region for the organisation of a stable disease-causing mutants (Ref. 90). Molecules sarcoglycan tetramer and association with that interfere with the ER quality control dystroglycan (Refs 22, 23). system, ERAD processing and ubiquitin One third of the a-sarcoglycan missense proteasome degradation may result in a greater mutations map to the cadherin-like domain, level of mutated sarcoglycan proteins, which which is believed to be important for Ca2þ- increases their chance of reaching the cell dependent association with the similar domain membrane. An essential prerequisite of this present in a-dystroglycan (Ref. 76). R34C, R34H, salvage approach is that the rescued protein D97G, R98S, R98C and R98H missense mutant maintains residual activity and/or mutations map to putative Ca2þ-binding sites in function at the cell membrane. It is thus the cadherin-like domain (Ref. 76). Substitution essential to first define the criteria for the in each of these three critical residues (R34, D97 identification of treatable mutants. Diverse and R98) causes LGMD in homozygous patients computational tools, based on both protein (Refs 44, 46). R74 causes a severe phenotype in a structural and evolutionary information, are homozygous patient (Ref. 54). Intriguingly, currently available to facilitate the prediction substitution of D97 and R98 residues seems to of the functional consequences of missense produce processing mutants, because they can mutations. In general, these methods give be pharmacologically rescued at the cell clues about whether replaced residues are membrane (Ref. 14). The R77C missense associated with a disease or are simply neutral substitution in the cadherin-like domain is the polymorphisms. Adverse mutations can either most frequently reported a-sarcoglycan destabilise the structure or disrupt a functional mutation. Analysis of muscle samples from site, such as ligand binding, catalytic or protein– homozygous R77C LGMD patients shows that protein interaction sites. a-sarcoglycan is absent in the majority of cases, Unfortunately, structural data on the sarcoglycan with variable residual expression of the sole complex are incomplete, so predictive approaches g-sarcoglycan (Refs 9, 44, 46, 47). Protein have to compensate for the lack of both expression data, however, do not tell us whether experimental data and identification of mutations within the cadherin-like domain also

homologous structures. In almost all cases of compromise a-sarcoglycan function. Sarcoglycanopathies: molecular pathogenesis and therapeutic prospects sarcoglycanopathy analysed so far, the mutants It is worth noting that seven a-sarcoglycan maybeconsideredtobeprocessingmutants. missense mutations cause the substitution of 12 Accession information: doi:10.1017/S1462399409001203; Vol. 11; e28; September 2009 & Cambridge University Press 2009. Re-use permitted under a Creative Commons Licence–by-nc-sa. expert reviews http://www.expertreviews.org/ in molecular medicine

α-sarcoglycan 1 MAETLFWTPL LVVLLAGLGD TEAQQTTLHPLVGRVFVHTL DHETFLSLPE HVAVPPAVHI 61 TYHAHLQGHP DLPRWLRYTQ RSPHHPGFLY GSATPEDRGL QVIEVTAYNR DSFDTTRQRL 121 VLEIGDPEGP LLPYQAEFLV RSHDAEEVLP STPASRFLSA LGGLWEPGEL QLLNVTSALD 181 RGGRVPLPIE GRKEGVYIKV GSASPFSTCL KMVASPDSHA RCAQGQPPLL SCYDTLAPHF 241 RVDWCNVTLV DKSVPEPADE VPTPGDGILE HDPFFCPPTE APDRDFLVDA LVTLLVPLLV 301 ALLLTLLLAY VMCCRREGRL KRDLATSDIQ MVHHCTIHGN TEELRQMAAS REVPRPLSTL 361 PMFNVHTGER LPPRVDSAQV PLILDQH

β-sarcoglycan 1 MAAAAAAAAE QQSSNGPVKK SMREKAVERR SVNKEHNSNF KAGYIPIDED RLHKTGLRGR 61 KGNLAICVII LLFILAVINL IITLVIWAVI RIGPNGCDSM EFHESGLLRF KQVSDMGVIH 121 PLYKSTVGGR RNENLVITGN NQPIVFQQGT TKLSVENNKT SITSDIGMQF FDPRTQNILF 181 STDYETHEFH LPSGVKSLNV QKASTERITS NATSDLNIKV DGRAIVRGNE GVFIMGKTIE 241 FHMGGNMELK AENSIILNGS VMVSTTRLPS SSSGDQLGSG DWVRYKLCMC ADGTLFKVQV 301 TSQNMGCQIS DNPCGNTH

γ-sarcoglycan 1 MVREQYTTAT EGICIERPEN QYVYKIGIYG WRKRCLYLFV LLLLIILVVN LALTIWILKV 61 MWFSPAGMGH LCVTKDGLRL EGESEFLFPL YAKEIHSRVD SSLLLQSTQN VTVNARNSEG 121 EVTGRLKVGP KMVEVQNQQF QINSNDGKPL FTVDEKEVVV GTDKLRVTGP EGALFEHSVE 181 TPLVRADPFQ DLRLESPTRS LSMDAPRGVH IQAHAGKIEA LSQMDILFHS SDGMLVLDAE 241 TVCLPKLVQG TWGPSGSSQS LYEICVCPDG KLYLSVAGVS TTCQEHSHIC L

δ-sarcoglycan 1 MMPQEQYTHH RSTMPGSVGP QVYKVGIYGW RKRCLYFFVL LLMILILVNL AMTIWILKVM 61 NFTIDGMGNL RITEKGLKLE GDSEFLQPLY AKEIQSRPGN ALYFKSARNV TVNILNDQTK 121 VLTQLITGPK AVEAYGKKFE VKTVSGKLLF SADNNEVVVG AERLRVLGAE GTVFPKSIET 181 PNVRADPFKE LRLESPTRSL VMEAPKGVEI NAEAGNMEAT CRTELRLESK DGEIKLDAAK 241 IRLPRLPHGS YTPTGTRQKV FEICVCANGR LFLSQAGAGS TCQINTSVCL

Sarcoglycan missense mutations Expert Reviews in Molecular Medicine © Cambridge University Press 2009

Figure 5. Sarcoglycan missense mutations. Sequence of a-, b-, g and d-sarcoglycan with all missense substitutions responsible for LGMD indicated in red. The indicated missense mutations are derived from the Leiden database (http://www.lovd.org) and recent reports (Refs 53, 92). The stretch of residues of the transmembrane domain is shown in yellow, the putative N-linked glycosylation sites in green, and the predicted phosphorylation sites (Ref. 95) in dark green. In a-sarcoglycan, the signal sequence is indicated in italics, the cadherin-like domain (Ref. 76) in grey, whereas the putative ATP-binding site (Ref. 38) is shown in blue. In b-, g- and d-sarcoglycan, the putative EGF-like sequence (Ref. 3) is underlined. diverse residues with a cysteine, which are expected (174NVT and 246NVT become, respectively, to cause abnormal disulphide bonds and abnormal 174NAT and 246NMT), which are conservative conformation of the nascent protein. However, it substitutions that usually do not affect has been demonstrated in a heterologous cell glycosylation. Nevertheless, homozygous V247M system that it is the replacement of a positive causes LGMD (Refs 46, 48), suggesting that residue at position 77, rather than the introduction they are processing mutants. Interestingly, five of a cysteine, that causes retention of the mutant additional a-sarcoglycan missense mutations protein (Ref. 12). This finding highlights just reside in the highly conserved putative how complicated the prediction of pathological a-sarcoglycan ATP-binding domain (Ref. 38, blue consequences associated with single mutations is box in Fig. 5). One of these, R221H, targets a

in proteins of undetermined function. critical residue in the ATP-binding pocket of Sarcoglycanopathies: molecular pathogenesis and therapeutic prospects Two mutations, V175A and V247M, map within the protein and causes a severe reduction of the N-glycosylation sequence of a-sarcoglycan a-sarcoglycan, with a slight reduction of the other 13 Accession information: doi:10.1017/S1462399409001203; Vol. 11; e28; September 2009 & Cambridge University Press 2009. Re-use permitted under a Creative Commons Licence–by-nc-sa. expert reviews http://www.expertreviews.org/ in molecular medicine subunits (Ref. 96). Thus, R221H probably causes a Mutations in g-sarcoglycan loss of function, whereas substitutions in the Forty mutations are described in the g-sarcoglycan remaining four residues did not affect the gene (SGCG), 16 missense mutations generating ATP-binding capacity. Nonetheless, the C232S a full protein with a single residue substitution mutation causes a severe LGMD in homozygous (Fig. 5) and 24 that generate a truncated protein patients (Ref. 53). or no protein at all. Three portions of the g- A single a-sarcoglycan mutation, M312R, sarcoglycan extracellular domain display possible maps in the intracellular domain, apparently critical functions, two for the assembly with without having adverse consequences on either b-sarcoglycan or a-sarcoglycan, and the the extracellular domain; nevertheless, this putative EGF-like domain. Residues 60–155 mutation causes LGMD in a homozygous in the g-sarcoglycan portion proximal to the patient (Ref. 97). Similarly, two mutations transmembrane domain are thought to be caused by deletions generate two a-sarcoglycan essential for the interaction with a-andb- proteins with normal extracellular domains sarcoglycan (Ref. 29). Although the relevant but with truncated, scrambled intracellular residues for binding to the sarcoglycan partners tails. At present, no data regarding the function are not known, it is expected that some of the of the a-sarcoglycan intracellular tail are missense mutations residing in this region available. It is therefore not clear whether compromise the correct assembly of the complex. these mutations generate a misfolded protein The homozygous Q82R mutation actually causes intercepted by the ERAD-C machinery or severe LGMD and the almost complete absence of whether they also abrogate unknown functions the four sarcoglycans from the cell membrane located therein. (Ref.53).However,again,someofthesemight simply be processing mutants. In CHO cells, Mutations in b-sarcoglycan mutations producing a truncated g-sarcoglycan Forty-nine mutations have been so far described without the cysteines in the EGF-like domain do in the b-sarcoglycan gene (SGCB), 29 of which not permit sarcoglycan complex assembly are missense mutations that generate a full (Ref. 82), as demonstrated in muscle samples from protein with a single residue substitution patients (Ref. 98). By contrast, in two LGMD-2C (Fig. 5). Removal of glycosylation sites in patients, a homozygous D525T mutation generates b-sarcoglycan is known to affect the assembly atruncatedg-sarcoglycan protein without the of the complex (Ref. 85). The bd-sarcoglycan EGF-like domain, which is apparently able to core is suggested to link dystrophin through assemble with the other sarcoglycans (Ref. 45). undefined portions of their intracellular tail The C283Y missense mutation in the (Ref. 29); five missense mutations reside in the g-sarcoglycan cysteine-rich domain could be b-sarcoglycan tail and might have either loss- functionally relevant, because this cysteine is of-function consequences or simply generate crucial in the EGF-like domain (Ref. 98). In fact, processing mutants; two of these (Q11G and C283Y can cause severe LGMD and it severely G56R) cause LGMD in homozygous patients. perturbs the assembly of a-sarcoglycan to the In the large extracellular domain, the portion of complex in COS-1 cells (Ref. 85). Intriguingly, the b-sarcoglycan proximal to the transmembrane L53P mutation in the transmembrane domain domain is thought to be required for the binding causes mild LGMD with the complete absence of to d-sarcoglycan (Ref. 29); nine missense the protein but traces of the other sarcoglycans mutations reside in this portion of b-sarcoglycan (Ref. 53). and may thus perturb the assembly of the sarcoglycan complex. Of these nine mutations, Mutations in d-sarcoglycan seven cause LGMD in homozygous patients Fourteen d-sarcoglycan gene (SGCD) mutations (http://www.lovd.org). Two additional missense have been so far found to cause LGMD-2F. mutations reside in apparently noncritical Eight are missense substitution and all reside in residues of the b-sarcoglycan cysteine-rich the extracellular portion (Fig. 5). The portion domain (N304S and D311N), which is a putative in the extracellular domain between residues

EGF-like sequence, and might not be functionally 57 and 92 of d-sarcoglycan (proximal to the Sarcoglycanopathies: molecular pathogenesis and therapeutic prospects relevant, even though homozygous N304S causes transmembrane domain) was shown to be amildphenotype. critical for the association with b-sarcoglycan 14 Accession information: doi:10.1017/S1462399409001203; Vol. 11; e28; September 2009 & Cambridge University Press 2009. Re-use permitted under a Creative Commons Licence–by-nc-sa. expert reviews http://www.expertreviews.org/ in molecular medicine

(Ref. 29). Three disease-causing mutations are aided by the small size of the cDNA. The within this portion. Considering the critical role most effective gene delivery was obtained of the bd-sarcoglycan core in the assembly of with nonpathogenic adeno-associated viruses sarcoglycans, these substitutions are expected to (reviewed in Ref. 100). Importantly, in animal have functional consequences. Similarly, the models of sarcoglycanopathies, the best results portion between residues 92 and 200 is believed were seen when the vector was injected before to be important for d-tog-sarcoglycan the onset of the pathology (Refs 102, 103, 104). interaction (Ref. 29). One substitution, R198P, However, to be effective in patients, gene resides in a recently identified highly conserved therapy must overcome critical issues, such as sequence (L190RLESPTRSL) (Ref. 29). As highly targeting the largest tissue of the body and low conserved sequences are predicted to be distribution of the vector, as well as the immune functionally important, it is not surprising that response (Ref. 101). homozygous expression causes a DMD-like Graftingofgeneticallymodifiedorhealthydonor phenotype (Refs 7, 48). Five d-sarcoglycan cellsisanotherapproachtotreatdystrophicmuscle. mutations generate a protein with a truncated Several stem cell populations exhibit myogenic extracellular domain and cause clinical potential and have been analysed for their ability phenotypes from LGMD to DMD-like in to correct the dystrophic phenotype. These homozygous patients. include muscle-derived satellite cells, bone- To date, there is not sufficient information to marrow-derived mesenchymal stem cells, muscle precisely predict the number of mutations in side-population cells, and cells derived from the four sarcoglycans that cause loss-of-function blood vessel walls, such as mesangioblasts or consequences or which result in processing pericytes (reviewed by Refs 99, 105). Although mutants. We estimate that about half of LGMD- a pluripotent bone-marrow-derived side causing missense mutations in the four population of stem cells failed to deliver d- sarcoglycans are potentially recoverable to the cell sarcoglycan (Ref. 106), muscle satellite cells have membrane, by preventing their premature been more successful (Ref. 107). Mesangioblasts degradation through pharmacological interference injected into muscles of a-sarcoglycan-null mice with the ERAD machinery. This percentage could and dystrophin-deficient dogs show potential for be even higher if the residues substituted within gene delivery (Refs 108, 109). In addition to the supposedly functional sarcoglycan regions do not difficulty of treating the most abundant tissue of affect that function. To this end, studies in the body, stem cell therapy presents several heterologous cell systems could facilitate the potential disadvantages, such as the requirement identification of critical residues of sarcoglycan for immunosuppression, difficulty in functional domains. Heterologous cells could be identification of suitable adult stem cells, and used in preliminary tests for the identification of possible diffusion and colonisation efficiencies. appropriate molecules and/or interventions that Last, but not least, caution must be used in would favour the recovery of mutant sarcoglycans. transferring the encouraging results obtained in animal models to humans. Therapeutic approaches for Pharmacological strategies are becoming an sarcoglycanopathies important alternative to gene delivery, because Sarcoglycanopathies are autosomal recessive drugs can more easily target each tissue of the disorders. The majority of LGMD cases are due body. In general, pharmacological therapies can to compound heterozygous conditions and only be directed either to circumvent the primary a small percentage result from homozygous defect or to treat its pathological consequences. mutations. This situation could be advantageous Recent evidence indicates that this approach can to some extent, because even partial restoration also be used to facilitate the processing of of a functional protein might correct the misfolded but functional protein mutants. The dystrophic phenotype. Two therapeutic first treatment to produce beneficial effects was approaches are currently being investigated for the use of corticosteroids in DMD; however, delivery of healthy genes into dystrophic besides the anti-inflammatory effect, the

muscles: gene and cell therapy (Refs 99, 100, mechanisms of action remain undefined. Another Sarcoglycanopathies: molecular pathogenesis and therapeutic prospects 101). Gene therapy has been carried out in all pharmaceutical approach successfully overcame animal models of sarcoglycanopathies, and is premature stop codons in mutated mRNAs, thus 15 Accession information: doi:10.1017/S1462399409001203; Vol. 11; e28; September 2009 & Cambridge University Press 2009. Re-use permitted under a Creative Commons Licence–by-nc-sa. expert reviews http://www.expertreviews.org/ in molecular medicine favouring translational read-through (Refs 101, considered to be processing mutants. Therefore 110). Initially, this approach used aminoglycoside the possibility to intervene in the different antibiotics, which successfully rescued steps of the cell quality control system offers dystrophin in mdx mice (Ref. 111) and was the great opportunities for therapeutic intervention. object of clinical trials in DMD patients. However, Considering that the majority of LGMD patients additional work in mdx mice demonstrated are compound heterozygotes, it is reasonable to divergent results (Ref. 112). Recently, PTC124, a expect that rescue of just one of the two allele very promising nonaminoglycoside agent, was products would ameliorate the dystrophic demonstrated to selectively induce ribosomal phenotype. read-through of premature stop codons but not Chemical and pharmacological chaperones the normal stop codon (Refs 113, 114). The use of can assist processing mutants by stabilising the specific antisense oligonucleotides to induce exon defective protein. Chemical chaperones create skipping and generate in-frame transcripts a more favourable environment for protein translated into functional, albeit smaller, protein mutant processing and trafficking. Typical could be an additional approach for the therapy chemical chaperones are small molecules (e.g. of diseases caused by mutations generating glycerol, dimethyl sulfoxide, 4-phenylbutyrate, premature stop codons. This approach is useful trimethylamine) that stabilise the folding for large proteins possessing many repeated process of mutant proteins and/or the assembly functional units (e.g. dystrophin spectrin-like of multimeric complexes by increasing repeats), some of which can be deleted without hydration of the folding mutant (Ref. 90). dramatic functional consequences (Refs 101, However, to be effective, these molecules must 115). This option is not applicable to be used at very high concentrations, a condition sarcoglycanopathies because there are no that rarely permits their utilisation in clinical dispensable domains in the extracellular portion trials. of sarcoglycans and the genomic structure of the Numerous molecules have been proposed as sarcoglycan genes prevents any meaningful exon pharmacological chaperones, which might be skipping. more useful than chemical chaperones because Alternative strategies aim to compensate for the they act in a more specific manner, are less toxic muscle mass wasting or to directly cope with the and are utilised at lower levels. An ideal pathological mechanisms in muscular dystrophy pharmacological chaperone is a molecule (reviewed by Ref. 100). However, contrasting that interacts with the functional sites of the results were obtained by targeting the activity of mutant, stabilising the protein until it regulators of muscle growth. For example, reaches the cell membrane. For example, treatment with insulin-like growth factor-1 (IGF-1) ligands of cell membrane receptors have been improved life expectancy of the d-sarcoglycan- shown to stabilise and facilitate their processing deficient hamster (Ref. 116) and beneficial effects (reviewed by Ref. 90). b-, g- and d-sarcoglycan were obtained by treating a-sarcoglycan-deficient possess an EGF-like domain in their mice with a deacetylase inhibitor that induced extracellular portion (Ref. 3), although the the expression of the myostatin inhibitor follistatin putative ligand modulating this receptor-like in satellite cells (Ref. 117). By contrast, function is unknown. Identification of this administration of inactive myostatin ameliorated factor would reveal a possible crucial function the dystrophic phenotype of calpain 3 but not of of the sarcoglycan complex and provide a a-sarcoglycan-null mice (Ref. 118). Similarly, candidate drug to promote the processing of the antibody-mediated inhibition of myostatin in sarcoglycan complex. g-sarcoglycan-deficient mice was ineffective As illustrated above, there are additional sites (Ref. 119). Additional work is thus necessary to for intervention to promote the processing of explore this important therapeutic option for potentially functional sarcoglycan mutants. In sarcoglycanopathies. addition to a general intervention aimed at modification of the ER environment towards Possible pharmacological treatment more favourable conditions (produced by

of sarcoglycan-processing mutants molecular chaperones that affect oxidative state Sarcoglycanopathies: molecular pathogenesis and therapeutic prospects As indicated above, all the sarcoglycan mutants or calcium levels), other more specific actions produced by single-residue substitutions can be can be envisaged. The eight steps indicated in 16 Accession information: doi:10.1017/S1462399409001203; Vol. 11; e28; September 2009 & Cambridge University Press 2009. Re-use permitted under a Creative Commons Licence–by-nc-sa. expert reviews http://www.expertreviews.org/ in molecular medicine

Figure 4 are all suitable targets for pharmaceutical (Ref. 12). Before directing terminally misfolded intervention. The retrograde beneficial effects protein to retrotranslocation to the cytosol, ERAD produced by inhibiting the final steps of the substrates are reduced by protein disulfide proteasome degradation process (Step 8) suggest isomerases (PDIs), such as the oxidoreductase that intervention in the intermediate steps of the ERp57. This action probably requires the ERAD pathway might also favour a reduction of assistance of BiP (GRP78). The ER surveillance sarcoglycan mutant disposal, permitting their apparatus can also detect redundant monomers assembly into a complete tetramer, and trafficking of unassembled multiprotein complexes, which to the cell membrane. Below, we provide a may be the case for sarcoglycans, causing the concise description of the ERAD–ubiquitin– degradation of correctly folded proteins, whose proteasome pathway. recognition as ERAD substrates remains an The ERAD process can be split into three basic intriguing process. parts: (1) initial recognition of the ERAD Subsequently, a composite lumenal and/or substrate; (2) targeting and retrotranslocation of membrane-associated surveillance system the ERAD substrate from the ER into the targets ERAD substrates for retrotranslocation cytosol; and (3) ubiquitination and degradation and degradation. Sarcoglycans defective in the of the ERAD substrate into small peptides extracellular domain, which represent the vast within the 20S proteasome core (Fig. 4) (Refs 11, majority of cases, are probably recognised by 12, 120). Sarcoglycans mature in the ER the ER lectins OS-9 and XTP3-B and the membrane, and are processed by distinct chaperones BiP and GRP94 (Fig. 4, Step 2) pathways of the quality control machinery, (Ref. 122), and then recruited to a according to whether the defects reside in transmembrane complex that operates the cytosolic, transmembrane or extracellular retrotranslocation process. This complex is (lumenal) domains (Refs 11, 120, 121). Three formed by SEL1 and HRD1, with SEL1 facing distinct ERAD pathways are in fact used the lumenal side and HRD1, an E3 ubiquitin according to the topology of the misfolded ligase, the cytosolic ER side (Ref. 120) (Fig. 4, protein. ER-soluble lumenal proteins and integral Step 3). The presence of a ubiquitin ligase membrane proteins with misfolded extracellular (HRD1) in this complex indicates that (lumenal) domains are degraded via the ERAD-L ubiquitination is strictly associated with pathway, membrane proteins with cytosolic translocation of ERAD substrates (Ref. 120). defects are degraded by the ERAD-C pathway, Ubiquitination of proteins is in fact a whereas those with misfolded transmembrane prerequisite for their degradation. This process domains are degraded by the ERAD-M pathway. is operated by the concerted and sequential As most of sarcoglycans mutations reside in the action of three families of enzymes: E1 and E2 extracellular domain, ERAD-L is almost certainly enzymes that respectively activate and used for their degradation. Since some patients conjugate ubiquitin to E3 enzymes, which have defects in the intracellular portion of eventually ligate ubiquitin to lysine-competent a-andb-sarcoglycan, cytosolic ER-associated residues in the ERAD substrate (Fig. 4, Step 4). components of the ERAD-C pathway could Sec61, which controls cotranslational entry of instead be involved in processing of these nascent proteins, is probably the channel mutants (Ref. 120). protein through which luminal misfolded Exit of misfolded proteins from the calnexin– proteins pass from the ER to the cytosol. In calreticulin cycle is determined by the ‘inactivity’ addition, Derlin proteins have been found in of UDP-glucose:glycoprotein glucosyltransferase- the retrotranslocation machinery, and possibly 1 (UGGT) (Fig. 3). Mannosidase I is a key form a pore themselves (Ref. 123). enzyme in directing misfolded proteins to A cytosolic complex comprising the ATPase p97, ERAD; by trimming terminal mannoses, UFD1 and NPL4 is at the crossroads of all three mannosidase I inhibits glucose readdition and ERAD pathways, with p97 providing the energy thus re-entry into the calnexin–calreticulin cycle. for retrotranslocation of monoubiquitinated In this initial recognition function, mannosidase ERAD substrates (Refs 120, 124). The

I is assisted by a family of mannosidase-like p97–UFD1–NPL4 complex, associated with the Sarcoglycanopathies: molecular pathogenesis and therapeutic prospects proteins, EDEM1–EDEM3, which operate both ER membrane via Derlin proteins (Fig. 4, Step 5), as mannosidases and chaperones (Fig. 4, Step 1) escorts the polyubiquitinated mutant protein 17 Accession information: doi:10.1017/S1462399409001203; Vol. 11; e28; September 2009 & Cambridge University Press 2009. Re-use permitted under a Creative Commons Licence–by-nc-sa. expert reviews http://www.expertreviews.org/ in molecular medicine destined to degradation toward the proteasome transmembrane proteins, such as a-sarcoglycan, (Ref. 120). Next, the polyubiquitinated protein is and proteins that possess EGF-like domains, deglycosylated by the cytosolic enzyme such as b-, g- and d-sarcoglycan (Ref. 127). peptide:N-glycanase (PNGase) (Fig. 4, Step 6), ERp57 or an analogous ER component could deubiquitinated (Fig. 4, Step 7) and finally thus be involved in the processing of degraded in the 20S proteasome core (Ref. 125) sarcoglycans and would be a possible target for (Fig. 4, Step 8). therapy. Interestingly, some antibiotics have It is important to mention that accumulation of been recently described to inhibit misfolded proteins in the ER lumen may elicit the oxidoreductase activity of ERp57 (Ref. 128). unfolded protein response (UPR) signal Biochemical and gene-silencing approaches have transduction pathway (Ref. 126). The function of identified many of the ERAD components the UPR is to quickly remove accumulated indicated in Fig. 4, but have also demonstrated aberrant substrates, which are frequently in the that modification of their activity might have form of aggregates, and restore the ER to an substantial consequences on the fate of efficiently operating maturation compartment. processing mutants. The first and most widely Activation of the UPR causes (1) upregulation of studied ERAD substrate is the cystic fibrosis ER-resident protein expression to facilitate the transmembrane regulator (CFTR), a cell proper folding of misfolded proteins; (2) reduced membrane chloride channel whose mutations are activity of the secretory pathway by decreasing responsible for cystic fibrosis (Refs 90, 129, 130). the expression of secretory cargo; (3) increased DF508 is the most frequent mutation of CFTR degradation of terminally misfolded proteins; that causes misfolding of a partially functional and (4) restrained translational initiation until protein. Similarly to a-sarcoglycan mutants, the abnormality has been minimised. However, proteasome inhibition (Fig. 4, Step 8) permitted long-lasting activation of the UPR might have the partial rescue of DF508 CFTR (Refs 131, 132). severe consequences for cell survival (Ref. 126). Miglustat (N-butyldeoxynojirimycin), an inhibitor Therefore, prolonged interference with the of a-1,2 glucosidase (Fig. 3b), effectively corrected ERAD pathway should be considered cautiously. the DF508 and G622D CFTR mutants in nasal In summary, we propose that misfolded or epithelial cells through an action that interferes truncated sarcoglycans are intercepted by the with the progress of the misfolded protein in ER quality control system, which sequentially the calnexin–calreticulin cycle (Ref. 131). activates ERAD, ubiquitin–proteasome Geldanamycin treatment in vitro causes the degradation and possibly UPR, to rapidly release of CFTR protein from competent remove defective proteins and to maintain an chaperones and the concomitant termination of efficient ER. Table 2 lists potential molecular ubiquitination and formation of a stable protein targets for therapeutical intervention within the (Ref. 133). Derlin-1 (Fig. 4, Step 5) is thought to diverse components of the ERAD machinery recognise misfolded nonubiquitinated CFTR, and that could promote the rescue of misfolded reduction of its expression led to significantly sarcoglycan mutants. To this end, it would also increased levels of DF508 CFTR (Ref. 134). The be important to determine whether the p97 ATPase (Fig. 4, Step 5) is also a potential sarcoglycan complex is specifically targeted in target for pharmacological therapy of ERAD degradation; the identification of muscle- sarcoglycanopathies, because downregulation of specific quality control or ERAD factors that p97 partly rescued DF508 CFTR (Ref. 132). participate in the degradation process of However, a similar approach must be considered sarcoglycan mutants would be of a great help very cautiously in skeletal muscle, because in the development of novel therapeutical mutations affecting p97 activity are responsible approaches. For example, although for inclusion body myopathy, which is glycoproteins are bound to calnexin and/or characterised by excessive accumulation of calreticulin, disulphide bond formation and ubiquitinated proteins (Ref. 135). Conversely, a folding are promoted by the recruitment of novel inhibitor of p97, Eeyarestatin I, was shown ERp57, a member of the protein disulphide to efficiently block degradation of ERAD

isomerase family. ERp57 is essential for efficient substrates (Ref. 136). N-glycanase is the sole Sarcoglycanopathies: molecular pathogenesis and therapeutic prospects folding of glycoproteins sharing common deglycosylating enzyme directing misfolded structural domains, for instance Type 1 proteins to 26S proteasome (Fig. 4, Step 6). 18 Accession information: doi:10.1017/S1462399409001203; Vol. 11; e28; September 2009 & Cambridge University Press 2009. Re-use permitted under a Creative Commons Licence–by-nc-sa. expert reviews http://www.expertreviews.org/ in molecular medicine

Table 2. ER quality control and ERAD targets for possible therapy

Location Target Activity or function Drug ER quality control PDI Disulphide isomerase Ribostamycin ERp57 Oxidoreductase, isomerase Vancomycin UGGT Folding sensor Mannosidase I Removal of mannose from Kifunensine and protein N-glycans deoxyMR EDEM1-3 Mannosidase-like activity Lumenal and membrane- GRP94 Chaperone, folding Geldanamycin associated ERAD recognition assistant and targeting BiP Chaperone, folding assistant OS9 Lectin, substrate recognition XTP3-B Lectin, substrate recognition ERAD membrane-associated SEL1L ER component associated dislocating proteins with HRD1 HRD1 Transmembrane E3 Ubiquitin ligase Sec61p Possible retrotranslocon channel Derlins Component of retrotranslocon channel ERAD membrane associated E1 E1 ubiquitin-activating and cytosolic ubiquitin enzymes enzymes E2 E2 ubiquitin-conjugating enzymes E3, HRD1 E3 ubiquitin ligases E4 Polyubiquitin enzymes ER cytosol and membrane p97-UFD1- ERAD protein extraction associated NPL4 complex p97 ATPase providing energy for Eeyarestatin I substrate extraction Derlins ER anchor for p97–UFD1– NPL4 complex Proteasome N-glycanase Removal of N-glycans Chloroacetamidyl before degradation chitobiose DUB Removal of ubiquitin before degradation 20S Degradation of ERAD Bortezomib, MG132 proteasome substrate The components indicated in this table are derived from recent ERAD reviews (Refs 11, 12, 120, 123); for many components, the specific role has not yet been conclusively established. A partial list of molecules known to interfere with the activity of some ERAD components is also included: Eeyarestatin I (Ref. 136), geldanamycin (Ref. 130), kifunensine and deoxymannojirimycine (deoxyMR) (Ref. 13), ribostamycin, inhibitor of PDI chaperone activity (Ref. 139), vancomycin (Ref. 128), chloroacetamidyl chitobiose (Ref. 137). Abbreviations: BiP, immunoglobulin heavy chain binding protein (GRP78); DUB, deubiquitinating enzyme; ERAD, ER-associated protein degradation; PDI, protein disulfide isomerase; UGGT, UDP-glucose:glycoprotein glucosyltransferase-1. Sarcoglycanopathies: molecular pathogenesis and therapeutic prospects

19 Accession information: doi:10.1017/S1462399409001203; Vol. 11; e28; September 2009 & Cambridge University Press 2009. Re-use permitted under a Creative Commons Licence–by-nc-sa. expert reviews http://www.expertreviews.org/ in molecular medicine

Inhibition of N-glycanase, by very low doses of the mutants. Both currently used and novel drugs pan-caspase inhibitor z-VAD-fmk, or by the novel are emerging as suitable candidates for the inhibitor chloroacetamidyl chitobiose (Ref. 137), pharmaceutical treatment of sarcoglycanopathies, was shown to prevent deglycosylation and slow and research should concentrate in ERAD- down retrotranslocation of misfolded proteins modulating compounds with the aim of (Ref. 138). This section and Table 2 briefly identifying promising therapeutical approaches summarise some of the diverse approaches used for future clinical trials. for the treatment of CFTR processing mutants that could also be applied to sarcoglycans. Outstanding research questions Finally, it is important to note that proteasome Research on ERAD pathways and the proteasome inhibition has been successfully used to rescue the system is dynamic, and novel components are expression and localisation of components of the continuously being discovered. Research on DGC in skeletal muscle of mdx mice, and DMD muscle-specific ERAD components should be and BDM explants, an effect obtained by reducing encouraged and the discovery of sarcoglycan- the elevated dismantling rate of disassembled specific chaperones and/or enzymes specifically proteins, which is due to the absence of assisting their processing would offer new dystrophin (Refs 140, 141, 142). Recently, it has avenues for future therapy. It is hoped that been shown that 1-sarcoglycan mutants that cause many molecules will be identified as potential myoclonus–dystonia syndrome, are also candidates for the treatment of intercepted by ER quality control and degraded sarcoglycanopathies. To this end, however, by the ubiquitin–proteasome system (Ref. 143), preclinical studies are needed to validate their suggesting that interfering with the ERAD process effectiveness and possible unwanted could be beneficial, even with this syndrome. consequences. Preliminary studies in heterologous Last, but not least, recent evidence shows cell systems will be mandatory to test the that inhibition of the proteasome with the pharmacology and toxicity of potential candidates FDA-approved inhibitor bortezomib (Velcade) in preparation for clinical trials. successfully rescued the localisation of D97G a-sarcoglycan mutant in muscle explants from an Acknowledgements and funding LGMD-2D patient (Ref. 14). Additional studies are R.B. is funded by the Italian National Research certainly necessary, but current knowledge offers Council (C.N.R.) and the Association Francaise an exciting perspective for the pharmacological contre les Miopathies. D.S. is funded by an treatment of sarcoglycan processing mutants. Athenaeum Project of the University of Padova. The contribution of the Italian Space Agency is Clinical implications gratefully acknowledged. Comments from the It is worth reporting that no clinical trials are peer reviewers are greatly appreciated. underway for the pharmaceutical treatment of sarcoglycanopathies. However, recent research References shows some promise (Refs 13, 14), particularly 1 Blake, D.J. et al. (2002) Function and with sarcoglycan mutations tightly linked to the genetics of dystrophin and dystrophin-related ER quality control system. Evidence indicates proteins in muscle. Physiological Reviews 82, that interfering with the ERAD machinery may 291-329 be a relatively safe approach. Apparently, 2 Davies, K.E. and Nowak, K.J. (2006) Molecular inhibition of mannosidase I with kifunensine mechanisms of muscular dystrophies: old and new does not produce pathological consequences in players. Nature Reviews. Molecular Cell Biology 7, mice (Ref. 13). Moreover, inhibition of the 762-773 proteasome with bortezomib is already in Phase 3 Hack, A.A. et al. (2000) Sarcoglycans in muscular III clinical trials for the treatment of multiple dystrophy. Microscopy Research and Technique myeloma (http://www.velcade.com). Thus, both 48, 167-180 drugs have the potential for treating 4 Ozawa, E. et al. (2005) Molecular and cell biology of sarcoglycanopathy patients. In this review, we the sarcoglycan complex. Muscle and Nerve 32,

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Further reading, resources and contacts

Publications Straub, V. and Bushby, K. (2008) Therapeutic possibilities in the autosomal recessive limb-girdle muscular dystrophies. Neurotherapeutics 5, 619-626 This review describes an overview of potential novel therapeutical interventions in autosomal recessive limb-girdle muscular dystrophies. Muir, L.A. and Chamberlain, J.S. (2009) Emerging strategies for cell and gene therapy of the muscular dystrophies. Expert Reviews in Molecular Medicine 11, e8 This review describes novel therapeutical interventions developed to cure Duchenne muscular dystrophy and that can be extended to other dystrophies. Linde, L. and Kerem, B. (2008) Introducing sense into nonsense in treatments of human genetic diseases. Trends in Genetics 24, 552-563 This review deals with therapeutical approaches aimed at curing nonsense mutations creating a premature stop codon. Braun, S. (2008) Muscular gene transfer using nonviral vectors. Current Gene Therapy 8, 391-405 This review reports on recent progresses of plasmid delivery of genes to muscles by using non-viral vectors.

Websites An up-to-date list of allelic variants of sarcoglycan genes is available at the Leiden Open Variation Database (LOVD) gene homepage, created by Johan den Dunnen and colleagues at Leiden University Medical Center: http://www.dmd.nl/sgca_seqvar.html The Muscular Dystrophy Association, MDA, together with numerous international associates, provides information on muscular dystrophies and helpful resources for patients: http://www.mda.org/ Treat Neuromuscular Disorders (TREAT-NMD) provide ample information about muscular dystrophies and updates on new therapeutic developments: http://www.treat-nmd.eu/home.php The Center for Neuromuscular Diseases, CNMD, is dedicated to clinical trials and therapy: http://www.cnmd.ac.uk/index.html ClinicalTrialsGov offers a complete list of worldwide clinical trials on muscular dystrophies, and other medical conditions: http://clinicaltrials.gov/ct2/home Sarcoglycanopathies: molecular pathogenesis and therapeutic prospects

26 Accession information: doi:10.1017/S1462399409001203; Vol. 11; e28; September 2009 & Cambridge University Press 2009. Re-use permitted under a Creative Commons Licence–by-nc-sa. expert reviews http://www.expertreviews.org/ in molecular medicine

Features associated with this article

Figures Figure 1. The dystrophin–glycoprotein complex. Figure 2. Trafficking of the sarcoglycan complex. Figure 3. Putative ER processing of wild-type and mutant sarcoglycans. Figure 4. Putative progression of sarcoglycan mutants through the ER-associated degradation pathway. Figure 5. Sarcoglycan missense mutations. Tables Table 1. Human sarcoglycan protein data. Table 2. ER quality control and ERAD targets for possible therapy.

Citation details for this article

Dorianna Sandona` and Romeo Betto (2009) Sarcoglycanopathies: molecular pathogenesis and therapeutic prospects. Expert Rev. Mol. Med. Vol. 11, e28, September 2009, doi:10.1017/S1462399409001203 Sarcoglycanopathies: molecular pathogenesis and therapeutic prospects

27 Accession information: doi:10.1017/S1462399409001203; Vol. 11; e28; September 2009 & Cambridge University Press 2009. Re-use permitted under a Creative Commons Licence–by-nc-sa.