Autophagy induction rescues muscular dystrophy

Collagen VI is an extracellular matrix protein forming a microfibrillar network in the endomysium of skeletal muscles. In humans, mutations in any of the three genes coding for collagen VI cause several skeletal muscle diseases, including Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD). Collagen VI null (Col6a1^–/–) mice display a myopathic phenotype resembling that of BM and UCMD patients. Muscles lacking collagen VI are characterized by the presence of dilated sarcoplasmic reticulum and dysfunctional mitochondria, which triggers apoptosis and leads to muscle wasting. We have found that accumulation of abnormal organelles is due to an impairment of autophagy. Reactivation of the autophagic flux by either nutritional approaches or by pharmacological and genetics tools removes dysfunctional organelles and greatly ameliorates the dystrophic phenotype.     

New molecular mechanism for Ullrich congenital muscular dystrophy: a heterozygous in-frame deletion in the COL6A1 gene causes a severe phenotype

Recessive mutations in two of the three collagen VI genes, COL6A2 and COL6A3, have recently been shown to cause Ullrich congenital muscular dystrophy (UCMD), a frequently severe disorder characterized by congenital muscle weakness with joint contractures and coexisting distal joint hyperlaxity. Dominant mutations in all three collagen VI genes had previously been associated with the considerably milder Bethlem myopathy. Here we report that a de novo heterozygous deletion of the COL6A1 gene can also result in a severe phenotype of classical UCMD precluding ambulation. The internal gene deletion occurs near a minisatellite DNA sequence in intron 8 that removes 1.1 kb of genomic DNA encompassing exons 9 and 10. The resulting mutant chain contains a 33-amino acid deletion near the amino-terminus of the triple-helical domain but preserves a unique cysteine in the triple-helical domain important for dimer formation prior to secretion. Thus, dimer formation and secretion of abnormal tetramers can occur and exert a strong dominant negative effect on microfibrillar assembly, leading to a loss of normal localization of collagen VI in the basement membrane surrounding muscle fibers. Consistent with this mechanism was our analysis of a patient with a much milder phenotype, in whom we identified a previously described Bethlem myopathy heterozygous in-frame deletion of 18 amino acids somewhat downstream in the triple-helical domain, a result of exon 14 skipping in the COL6A1 gene. This deletion removes the crucial cysteine, so that dimer formation cannot occur and the abnormal molecule is not secreted, preventing the strong dominant negative effect. Our studies provide a biochemical insight into genotype-phenotype correlations in this group of disorders and establish that UCMD can be caused by dominantly acting mutations.     

Defective collagen VI α6 chain expression in the skeletal muscle of patients with collagen VI-related myopathies

Collagen VI is a non-fibrillar collagen present in the extracellular matrix (ECM) as a complex polymer; the mainly expressed form is composed of α1, α2 and α3 chains; mutations in genes encoding these chains cause myopathies known as Ullrich congenital muscular dystrophy (UCMD), Bethlem myopathy (BM) and myosclerosis myopathy (MM). The collagen VI α6 chain is a recently identified component of the ECM of the human skeletal muscle. Here we report that the α6 chain was dramatically reduced in skeletal muscle and muscle cell cultures of genetically characterized UCMD, BM and MM patients, independently of the clinical phenotype, the gene involved and the effect of the mutation on the expression of the “classical” α1α2α3 heterotrimer. By contrast, the collagen VI α6 chain was normally expressed or increased in the muscle of patients affected by other forms of muscular dystrophy, the overexpression matching with areas of increased fibrosis. In vitro treatment with TGF-β1, a potent collagen inducer, promoted the collagen VI α6 chain deposition in the ECM of normal muscle cells, whereas, in cultures derived from collagen VI-related myopathy patients, the collagen VI α6 chain failed to develop a network outside the cells and accumulated in the endoplasmic reticulum. The defect of the α6 chain points to a contribution to the pathogenesis of collagen VI-related disorders.     

Col6a1 Null Mice as a Model to Study Skin Phenotypes in Patients with Collagen VI Related Myopathies: Expression of Classical and Novel Collagen VI Variants during Wound Healing

Patients suffering from collagen VI related myopathies caused by mutations in COL6A1, COL6A2 and COL6A3 often also display skin abnormalities, like formation of keloids or “cigarette paper” scars, dry skin, striae rubrae and keratosis pilaris (follicular keratosis). Here we evaluated if Col6a1 null mice, an established animal model for the muscle changes in collagen VI related myopathies, are also suitable for the study of mechanisms leading to the skin pathology. We performed a comprehensive study of the expression of all six collagen VI chains in unwounded and challenged skin of wild type and Col6a1 null mice. Expression of collagen VI chains is regulated in both skin wounds and bleomycin-induced fibrosis and the collagen VI α3 chain is proteolytically processed in both wild type and Col6a1 null mice. Interestingly, we detected a decreased tensile strength of the skin and an altered collagen fibril and basement membrane architecture in Col6a1 null mice, the latter being features that are also found in collagen VI myopathy patients. Although Col6a1 null mice do not display an overt wound healing defect, these mice are a relevant animal model to study the skin pathology in collagen VI related disease.     

Aberrant Mitochondria in a Bethlem Myopathy Patient with a Homozygous Amino Acid Substitution That Destabilizes the Collagen VI α2(VI) Chain

Bethlem myopathy and Ullrich congenital muscular dystrophy (UCMD) sit at opposite ends of a clinical spectrum caused by mutations in the extracellular matrix protein collagen VI. Bethlem myopathy is relatively mild, and patients remain ambulant in adulthood while many UCMD patients lose ambulation by their teenage years and require respiratory interventions. Dominant and recessive mutations are found across the entire clinical spectrum; however, recessive Bethlem myopathy is rare, and our understanding of the molecular pathology is limited. We studied a patient with Bethlem myopathy. Electron microscopy of his muscle biopsy revealed abnormal mitochondria. We identified a homozygous COL6A2 p.D871N amino acid substitution in the C-terminal C2 A-domain. Mutant α2(VI) chains are unable to associate with α1(VI) and α3(VI) and are degraded by the proteasomal pathway. Some collagen VI is assembled, albeit more slowly than normal, and is secreted. These molecules contain the minor α2(VI) C2a splice form that has an alternative C terminus that does include the mutation. Collagen VI tetramers containing the α2(VI) C2a chain do not assemble efficiently into microfibrils and there is a severe collagen VI deficiency in the extracellular matrix. We expressed wild-type and mutant α2(VI) C2 domains in mammalian cells and showed that while wild-type C2 domains are efficiently secreted, the mutant p.D871N domain is retained in the cell. These studies shed new light on the protein domains important for intracellular and extracellular collagen VI assembly and emphasize the importance of molecular investigations for families with collagen VI disorders to ensure accurate diagnosis and genetic counseling.     

Autophagy induction rescues muscular dystrophy

Collagen VI is an extracellular matrix protein forming a microfibrillar network in the endomysium of skeletal muscles. In humans, mutations in any of the three genes coding for collagen VI cause several skeletal muscle diseases, including Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD). Collagen VI null (Col6a1(-/-)) mice display a myopathic phenotype resembling that of BM and UCMD patients. Muscles lacking collagen VI are characterized by the presence of dilated sarcoplasmic reticulum and dysfunctional mitochondria, which triggers apoptosis and leads to muscle wasting. We have found that accumulation of abnormal organelles is due to an impairment of autophagy. Reactivation of the autophagic flux by either nutritional approaches or by pharmacological and genetics tools removes dysfunctional organelles and greatly ameliorates the dystrophic phenotype.     

Effects on collagen VI mRNA stability and microfibrillar assembly of three COL6A2 mutations in two families with Ullrich congenital muscular dystrophy

We recently reported a severe deficiency in collagen type VI, resulting from recessive mutations of the COL6A2 gene, in patients with Ullrich congenital muscular dystrophy. Their parents, who are all carriers of one mutant allele, are unaffected, although heterozygous mutations in collagen VI caused Bethlem myopathy. Here we investigated the consequences of three COL6A2 mutations in fibroblasts from patients and their parents in two Ullrich families. All three mutations lead to nonsense-mediated mRNA decay. However, very low levels of undegraded mutant mRNA remained in patient B with compound heterozygous mutations at the distal part of the triple-helical domain, resulting in deposition of abnormal microfibrils that cannot form extensive networks. This observation suggests that the C-terminal globular domain is not essential for triple-helix formation but is critical for microfibrillar assembly. In all parents, the COL6A2 mRNA levels are reduced to 57-73% of the control, but long term collagen VI matrix depositions are comparable with that of the control. The almost complete absence of abnormal protein and near-normal accumulation of microfibrils in the parents may account for their lack of myopathic symptoms.     

Recessive COL6A2 C-globular Missense Mutations in Ullrich Congenital Muscular Dystrophy: ROLE OF THE C2a SPLICE VARIANT*

Ullrich congenital muscular dystrophy (UCMD) is a disabling and life-threatening disorder resulting from either recessive or dominant mutations in genes encoding collagen VI. Although the majority of the recessive UCMD cases have frameshift or nonsense mutations in COL6A1, COL6A2, or COL6A3, recessive structural mutations in the COL6A2 C-globular region are emerging also. However, the underlying molecular mechanisms have remained elusive. Here we identified a homozygous COL6A2 E624K mutation (C1 subdomain) and a homozygous COL6A2 R876S mutation (C2 subdomain) in two UCMD patients. The consequences of the mutations were investigated using fibroblasts from patients and cells stably transfected with the mutant constructs. In contrast to expectations based on the clinical severity of these two patients, secretion and assembly of collagen VI were moderately affected by the E624K mutation but severely impaired by the R876S substitution. The E624K substitution altered the electrostatic potential of the region surrounding the metal ion-dependent adhesion site, resulting in a collagen VI network containing thick fibrils and spots with densely packed microfibrils. The R876S mutation prevented the chain from assembling into triple-helical collagen VI molecules. The minute amount of collagen VI secreted by the R876S fibroblasts was solely composed of a faster migrating chain corresponding to the C2a splice variant with an alternative C2 subdomain. In transfected cells, the C2a splice variant was able to assemble into short microfibrils. Together, the results suggest that the C2a splice variant may functionally compensate for the loss of the normal COL6A2 chain when mutations occur in the C2 subdomain.     

Autophagy is defective in collagen VI muscular dystrophies, and its reactivation rescues myofiber degeneration

Autophagy is crucial in the turnover of cell components, and clearance of damaged organelles by the autophagic-lysosomal pathway is essential for tissue homeostasis. Defects of this degradative system have a role in various diseases, but little is known about autophagy in muscular dystrophies. We have previously found that muscular dystrophies linked to collagen VI deficiency show dysfunctional mitochondria and spontaneous apoptosis, leading to myofiber degeneration. Here we demonstrate that this persistence of abnormal organelles and apoptosis are caused by defective autophagy. Skeletal muscles of collagen VI-knockout (Col6a1(-/-)) mice had impaired autophagic flux, which matched the lower induction of beclin-1 and BCL-2/adenovirus E1B-interacting protein-3 (Bnip3) and the lack of autophagosomes after starvation. Forced activation of autophagy by genetic, dietary and pharmacological approaches restored myofiber survival and ameliorated the dystrophic phenotype of Col6a1(-/-) mice. Furthermore, muscle biopsies from subjects with Bethlem myopathy or Ullrich congenital muscular dystrophy had reduced protein amounts of beclin-1 and Bnip3. These findings indicate that defective activation of the autophagic machinery is pathogenic in some congenital muscular dystrophies.     

Collagen VI myopathies: From the animal model to the clinical trial

Collagen VI is an ECM protein which forms a prominent microfibrillar network in the endomysium of skeletal muscle. Mutations in the genes coding for the three chains of collagen VI cause skeletal muscle diseases; the severe wasting Ullrich congenital muscular dystrophy (UCMD) normally present at birth, and the milder Bethlem myopathy (BM). The pathogenesis of both collagen VI myopathies was unknown until 2003. Our group, utilizing Col6a1 deficient mice, discovered a latent mitochondrial dysfunction that caused increased apoptosis in muscle cells. These effects could be reverted by incubating Col6a1 null muscle fibres with cyclosporin A (CsA), an inhibitor of the mitochondrial permeability pore; more interestingly, the treatment of Col6a1 null mice with CoA rescued the muscle phenotype in vivo.These findings demonstrated an unexpected collagen VI/mitochondrial connection as the basis for the UCMD and BM pathogenesis and suggested a strategy for a possible pharmacological treatment of the diseases. This was assessed by demonstrating that muscle biopsies from patients with UCMD showed an abnormal mitochondrial depolarization and that treatment with CsA normalized the mitochondrial phenotype.In this study we report the results of an open pilot trial of four UCMD and one BM patients, representing a range of collagen VI deficiency and having mutations in three of the collagen VI genes. As determined in muscle biopsies prior to treatment, all patients displayed mitochondrial dysfunction and muscle fibres showed an increased frequency of apoptosis. When patients were treated for 1 month with a low daily dose of CsA, primary muscle cell cultures of biopsies obtained at the end of the treatment showed a decreased apoptosis and increased immunohistochemical signs of muscle fibre regeneration. These results confirm that the pathogenic mechanism found in Col6a1 deficient mice also plays a crucial role in hereditary muscle diseases in human, and suggest that targeted treatment of these mitochondrial defects in patients with UCMD and BM may be effective in preventing and/or reverting muscle alterations.It is also important to consider that desensitization of the permeability transition pore by CsA occurs independently of calcineurin inhibition; because a CsA derivative that has no immunosuppressive activity appears to be as effective as the parent molecule, long-term trials should be designed to prevent irreversible muscle damages in young patients affected by collagen VI myopathies, without exposing them to infective risks.     

Autophagy activation in COL6 myopathic patients by a low-protein-diet pilot trial

A pilot clinical trial based on nutritional modulation was designed to assess the efficacy of a one-year low-protein diet in activating autophagy in skeletal muscle of patients affected by COL6/collagen VI-related myopathies. Ullrich congenital muscular dystrophy and Bethlem myopathy are rare inherited muscle disorders caused by mutations of COL6 genes and for which no cure is yet available. Studies in col6 null mice revealed that myofiber degeneration involves autophagy defects and that forced activation of autophagy results in the amelioration of muscle pathology. Seven adult patients affected by COL6 myopathies underwent a controlled low-protein diet for 12 mo and we evaluated the presence of autophagosomes and the mRNA and protein levels for BECN1/Beclin 1 and MAP1LC3B/LC3B in muscle biopsies and blood leukocytes. Safety measures were assessed, including muscle strength, motor and respiratory function, and metabolic parameters. After one y of low-protein diet, autophagic markers were increased in skeletal muscle and blood leukocytes of patients. The treatment was safe as shown by preservation of lean:fat percentage of body composition, muscle strength and function. Moreover, the decreased incidence of myofiber apoptosis indicated benefits in muscle homeostasis, and the metabolic changes pointed at improved mitochondrial function. These data provide evidence that a low-protein diet is able to activate autophagy and is safe and tolerable in patients with COL6 myopathies, pointing at autophagy activation as a potential target for therapeutic applications. In addition, our findings indicate that blood leukocytes are a promising noninvasive tool for monitoring autophagy activation in patients.     

The effect of carbamazepine on bone structure and strength in control and osteogenesis imperfecta (Col1a2 +/p.G610C ) mice

The inherited brittle bone disease osteogenesis imperfecta (OI) is commonly caused by COL1A1 and COL1A2 mutations that disrupt the collagen I triple helix. This causes intracellular endoplasmic reticulum (ER) retention of the misfolded collagen and can result in a pathological ER stress response. A therapeutic approach to reduce this toxic mutant load could be to stimulate mutant collagen degradation by manipulating autophagy and/or ER‐associated degradation. Since carbamazepine (CBZ) both stimulates autophagy of misfolded collagen X and improves skeletal pathology in a metaphyseal chondrodysplasia model, we tested the effect of CBZ on bone structure and strength in 3‐week‐old male OI Col1a2 ^+/p.G610C and control mice. Treatment for 3 or 6 weeks with CBZ, at the dose effective in metaphyseal chondrodysplasia, provided no therapeutic benefit to Col1a2 ^+/p.G610C mouse bone structure, strength or composition, measured by micro‐computed tomography, three point bending tests and Fourier‐transform infrared microspectroscopy. In control mice, however, CBZ treatment for 6 weeks impaired femur growth and led to lower femoral cortical and trabecular bone mass. These data, showing the negative impact of CBZ treatment on the developing mouse bones, raise important issues which must be considered in any human clinical applications of CBZ in growing individuals.     

Three novel collagen VI chains with high homology to the alpha3 chain

Here we describe three novel collagen VI chains, alpha4, alpha5, and alpha6. The corresponding genes are arranged in tandem on mouse chromosome 9. The new chains structurally resemble the collagen VI alpha3 chain. Each chain consists of seven von Willebrand factor A domains followed by a collagenous domain, two C-terminal von Willebrand factor A domains, and a unique domain. In addition, the collagen VI alpha4 chain carries a Kunitz domain at the C terminus, whereas the collagen VI alpha5 chain contains an additional von Willebrand factor A domain and a unique domain. The size of the collagenous domains and the position of the structurally important cysteine residues within these domains are identical between the collagen VI alpha3, alpha4, alpha5, and alpha6 chains. In mouse, the new chains are found in or close to basement membranes. Collagen VI alpha1 chain-deficient mice lack expression of the new collagen VI chains implicating that the new chains may substitute for the alpha3 chain, probably forming alpha1alpha2alpha4, alpha1alpha2alpha5, or alpha1alpha2alpha6 heterotrimers. Due to a large scale pericentric inversion, the human COL6A4 gene on chromosome 3 was broken into two pieces and became a non-processed pseudogene. Recently COL6A5 was linked to atopic dermatitis and designated COL29A1. The identification of novel collagen VI chains carries implications for the etiology of atopic dermatitis as well as Bethlem myopathy and Ullrich congenital muscular dystrophy.     

Recessive Mutations in the α3 (VI) Collagen Gene COL6A3 Cause Early-Onset Isolated Dystonia

Isolated dystonia is a disorder characterized by involuntary twisting postures arising from sustained muscle contractions. Although autosomal-dominant mutations in TOR1A, THAP1, and GNAL have been found in some cases, the molecular mechanisms underlying isolated dystonia are largely unknown. In addition, although emphasis has been placed on dominant isolated dystonia, the disorder is also transmitted as a recessive trait, for which no mutations have been defined. Using whole-exome sequencing in a recessive isolated dystonia-affected kindred, we identified disease-segregating compound heterozygous mutations in COL6A3, a collagen VI gene associated previously with muscular dystrophy. Genetic screening of a further 367 isolated dystonia subjects revealed two additional recessive pedigrees harboring compound heterozygous mutations in COL6A3. Strikingly, all affected individuals had at least one pathogenic allele in exon 41, including an exon-skipping mutation that induced an in-frame deletion. We tested the hypothesis that disruption of this exon is pathognomonic for isolated dystonia by inducing a series of in-frame deletions in zebrafish embryos. Consistent with our human genetics data, suppression of the exon 41 ortholog caused deficits in axonal outgrowth, whereas suppression of other exons phenocopied collagen deposition mutants. All recessive mutation carriers demonstrated early-onset segmental isolated dystonia without muscular disease. Finally, we show that Col6a3 is expressed in neurons, with relevant mRNA levels detectable throughout the adult mouse brain. Taken together, our data indicate that loss-of-function mutations affecting a specific region of COL6A3 cause recessive isolated dystonia with underlying neurodevelopmental deficits and highlight the brain extracellular matrix as a contributor to dystonia pathogenesis.     

COL4A1 mutations cause ocular dysgenesis, neuronal localization defects, and myopathy in mice and Walker-Warburg syndrome in humans

Muscle-eye-brain disease (MEB) and Walker Warburg Syndrome (WWS) belong to a spectrum of autosomal recessive diseases characterized by ocular dysgenesis, neuronal migration defects, and congenital muscular dystrophy. Until now, the pathophysiology of MEB/WWS has been attributed to alteration in dystroglycan post-translational modification. Here, we provide evidence that mutations in a gene coding for a major basement membrane protein, collagen IV alpha 1 (COL4A1), are a novel cause of MEB/WWS. Using a combination of histological, molecular, and biochemical approaches, we show that heterozygous Col4a1 mutant mice have ocular dysgenesis, neuronal localization defects, and myopathy characteristic of MEB/WWS. Importantly, we identified putative heterozygous mutations in COL4A1 in two MEB/WWS patients. Both mutations occur within conserved amino acids of the triple-helix-forming domain of the protein, and at least one mutation interferes with secretion of the mutant proteins, resulting instead in intracellular accumulation. Expression and posttranslational modification of dystroglycan is unaltered in Col4a1 mutant mice indicating that COL4A1 mutations represent a distinct pathogenic mechanism underlying MEB/WWS. These findings implicate a novel gene and a novel mechanism in the etiology of MEB/WWS and expand the clinical spectrum of COL4A1-associated disorders.     

Ultrastructural defects of collagen VI filaments in an Ullrich syndrome patient with loss of the alpha3(VI) N10-N7 domains

Ultrastructural alterations of collagen VI in cultured fibroblasts and reduced collagen VI immunostaining in the papillary dermis and endomysium were detected in a patient with a mild form of Ullrich congenital muscular dystrophy caused by a COL6A3 gene mutation. The patient had been previously demonstrated to express an alpha3(VI) chain shorter than normal due to skipping of the mutated exon. We show that collagen VI filaments are not organized in a normal network in the extracellular matrix secreted by patient's cultured fibroblasts. Moreover, we demonstrate that in this patient the alpha3(VI) chain is produced in lower amounts and it is almost exclusively represented by the shorter, alternatively spliced N6-C5 isoform. These results suggest that different alpha3(VI) chain isoforms, containing also domains of the N10-N7 region, are required for assembling a proper collagen VI network in the extracellular matrix.     

Collagen VI Microfibril Formation Is Abolished by an α2(VI) von Willebrand Factor Type A Domain Mutation in a Patient with Ullrich Congenital Muscular Dystrophy

Collagen VI is an extracellular protein that most often contains the three genetically distinct polypeptide chains, α1(VI), α2(VI), and α3(VI), although three recently identified chains, α4(VI), α5(VI), and α6(VI), may replace α3(VI) in some situations. Each chain has a triple helix flanked by N- and C-terminal globular domains that share homology with the von Willebrand factor type A (VWA) domains. During biosynthesis, the three chains come together to form triple helical monomers, which then assemble into dimers and tetramers. Tetramers are secreted from the cell and align end-to-end to form microfibrils. The precise molecular mechanisms responsible for assembly are unclear. Mutations in the three collagen VI genes can disrupt collagen VI biosynthesis and matrix organization and are the cause of the inherited disorders Bethlem myopathy and Ullrich congenital muscular dystrophy. We have identified a Ullrich congenital muscular dystrophy patient with compound heterozygous mutations in α2(VI). The first mutation causes skipping of exon 24, and the mRNA is degraded by nonsense-mediated decay. The second mutation is a two-amino acid deletion in the C1 VWA domain. Recombinant C1 domains containing the deletion are insoluble and retained intracellularly, indicating that the mutation has detrimental effects on domain folding and structure. Despite this, mutant α2(VI) chains retain the ability to associate into monomers, dimers, and tetramers. However, we show that secreted mutant tetramers containing structurally abnormal C1 VWA domains are unable to associate further into microfibrils, directly demonstrating the critical importance of a correctly folded α2(VI) C1 domain in microfibril formation.