Immune-Mediated Rippling Muscle Disease Associated With Thymoma and Anti-MURC/Cavin-4 Autoantibodies.

OBJECTIVES: Rippling muscle disease (RMD) is characterized by muscle stiffness, muscle hypertrophy, and rippling muscle induced by stretching or percussion. Hereditary RMD is due to sequence variants in the CAV3 and PTRF/CAVIN1 genes encoding Caveolin-3 or Cavin-1, respectively; a few series of patients with acquired autoimmune forms of RMD (iRMD) associated with AChR antibody-positive myasthenia gravis and/or thymoma have also been described. Recently, MURC/caveolae-associated protein 4 (Cavin-4) autoantibody was identified in 8 of 10 patients without thymoma, highlighting its potential both as a biomarker and as a triggering agent of this pathology. Here, we report the case of a patient with iRMD-AchR antibody negative associated with thymoma. METHODS: We suspected a paraneoplastic origin and investigated the presence of specific autoantibodies targeting muscle antigens through a combination of Western blotting and affinity purification coupled with mass spectrometry-based proteomic approaches. RESULTS: We identified circulating MURC/Cavin-4 autoantibodies and found strong similarities between histologic features of the patient's muscle and those commonly reported in caveolinopathies. Strikingly, MURC/Cavin-4 autoantibody titer strongly decreased after tumor resection and immunotherapy correlating with complete disappearance of the rippling phenotype and full patient remission. DISCUSSION: MURC/Cavin-4 autoantibodies may play a pathogenic role in paraneoplastic iRMD associated with thymoma.

Differential physiological roles for BIN1 isoforms in skeletal muscle development, function and regeneration.

Skeletal muscle development and regeneration are tightly regulated processes. How the intracellular organization of muscle fibers is achieved during these steps is unclear. Here, we focus on the cellular and physiological roles of amphiphysin 2 (BIN1), a membrane remodeling protein mutated in both congenital and adult centronuclear myopathies (CNM), that is ubiquitously expressed and has skeletal muscle-specific isoforms. We created and characterized constitutive muscle-specific and inducible Bin1 homozygous and heterozygous knockout mice targeting either ubiquitous or muscle-specific isoforms. Constitutive Bin1-deficient mice died at birth from lack of feeding due to a skeletal muscle defect. T-tubules and other organelles were misplaced and altered, supporting a general early role for BIN1 in intracellular organization, in addition to membrane remodeling. Although restricted deletion of Bin1 in unchallenged adult muscles had no impact, the forced switch from the muscle-specific isoforms to the ubiquitous isoforms through deletion of the in-frame muscle-specific exon delayed muscle regeneration. Thus, ubiquitous BIN1 function is necessary for muscle development and function, whereas its muscle-specific isoforms fine tune muscle regeneration in adulthood, supporting that BIN1 CNM with congenital onset are due to developmental defects, whereas later onset may be due to regeneration defects.

Donskoy cats as a new model of oculocutaneous albinism with the identification of a splice-site variant in Hermansky-Pudlak Syndrome 5 gene.

In the feline Donskoy breed, a phenotype that breeders call "pink-eye," with associated light-brown skin, yellow irises and red-eye effect, has been described. Genealogical data indicated an autosomal recessive inheritance pattern. A single candidate region was identified by genome-wide association study and SNP-based homozygosity mapping. Within that region, we further identified HPS5 (HPS5 Biogenesis Of Lysosomal Organelles Complex 2 Subunit 2) as a strong candidate gene, since HPS5 variants have been identified in humans and animals with Hermansky-Pudlak syndrome 5 or oculocutaneous albinism. A homozygous c.2571-1G>A acceptor splice-site variant located in intron 16 of HPS5 was identified in pink-eye cats. Segregation of the variant was 100% consistent with the inheritance pattern. Genotyping of 170 cats from 19 breeds failed to identify a single carrier in non-Donskoy cats. The c.2571-1G>A variant leads to HPS5 exon-16 splicing that is predicted to produce a 52 amino acids in-frame deletion in the protein. These results support an association of the pink-eye phenotype with the c.2571-1G>A variant. The pink-eye Donskoy cat extends the panel of reported HPS5 variants and offers an opportunity for in-depth exploration of the phenotypic consequences of a new HPS5 variant.

Genetic heterogeneity of polydactyly in Maine Coon cats.

OBJECTIVES: Polydactyly has been described in two breeds of domestic cats (Maine Coon and Pixie Bob) and in some outbred domestic cats (eg, Hemingway cats). In most cases, feline polydactyly is a non-syndromic preaxial polydactyly. Three variants located in a regulatory sequence involved in limb development, named ZRS (zone of polarising activity regulatory sequence), have been identified to be responsible for feline polydactyly. These variants have been found in outbred domestic cats in the UK (UK1 and UK2 variants) and in Hemingway cats in the USA (Hw variant). The aim of this study was to characterise the genetic features of polydactyly in Maine Coon cats. METHODS: Genotyping assay was used to identify the variant(s) segregating in a cohort of 75 polydactyl and non-polydactyl Maine Coon cats from different breeding lines from Europe, Canada and the USA. The authors performed a segregation analysis to identify the inheritance pattern of polydactyly in this cohort and analysed the population structure. RESULTS: The Hw allele was identified in a subset of polydactyl cats. Sequencing of two regulatory sequences involved in limb development did not reveal any other variant in polydactyl cats lacking the Hw allele. Additionally, genotype-phenotype and segregation analyses revealed the peculiar inheritance pattern of polydactyly in Maine Coon cats. The population structure analysis demonstrated a genetic distinction between Hw and Hw-free polydactyl cats. CONCLUSIONS AND RELEVANCE: Polydactyly in Maine Coon cats is inherited as an autosomal dominant trait with incomplete penetrance and variable expressivity, and this trait is characterised by genetic heterogeneity in the Maine Coon breed. Maine Coon breeders should be aware of this situation and adapt their breeding practices accordingly.

Genetic Evidence That Captured Retroviral Envelope syncytins Contribute to Myoblast Fusion and Muscle Sexual Dimorphism in Mice.

Syncytins are envelope genes from endogenous retroviruses, "captured" for a role in placentation. They mediate cell-cell fusion, resulting in the formation of a syncytium (the syncytiotrophoblast) at the fetomaternal interface. These genes have been found in all placental mammals in which they have been searched for. Cell-cell fusion is also pivotal for muscle fiber formation and repair, where the myotubes are formed from the fusion of mononucleated myoblasts into large multinucleated structures. Here we show, taking advantage of mice knocked out for syncytins, that these captured genes contribute to myoblast fusion, with a >20% reduction in muscle mass, mean muscle fiber area and number of nuclei per fiber in knocked out mice for one of the two murine syncytin genes. Remarkably, this reduction is only observed in males, which subsequently show muscle quantitative traits more similar to those of females. In addition, we show that syncytins also contribute to muscle repair after cardiotoxin-induced injury, with again a male-specific effect on the rate and extent of regeneration. Finally, ex vivo experiments carried out on murine myoblasts demonstrate the direct involvement of syncytins in fusion, with a >40% reduction in fusion index upon addition of siRNA against both syncytins. Importantly, similar effects are observed with primary myoblasts from sheep, dog and human, with a 20-40% reduction upon addition of siRNA against the corresponding syncytins. Altogether, these results show a direct contribution of the fusogenic syncytins to myogenesis, with a demonstrated male-dependence of the effect in mice, suggesting that these captured genes could be responsible for the muscle sexual dimorphism observed in placental mammals.

Amphiphysin 2 Orchestrates Nucleus Positioning and Shape by Linking the Nuclear Envelope to the Actin and Microtubule Cytoskeleton.

Nucleus positioning is key for intracellular organization, cell differentiation, and organ development and is affected in many diseases, including myopathies due to alteration in amphiphysin-2 (BIN1). The actin and microtubule cytoskeletons are essential for nucleus positioning, but their crosstalk in this process is sparsely characterized. Here, we report that impairment of amphiphysin/BIN1 in Caenorhabditis elegans, mammalian cells, or muscles from patients with centronuclear myopathy alters nuclear position and shape. We show that AMPH-1/BIN1 binds to nesprin and actin, as well as to the microtubule-binding protein CLIP170 in both species. Expression of the microtubule-anchoring CAP-GLY domain of CLIP170 fused to the nuclear-envelope-anchoring KASH domain of nesprin rescues nuclear positioning defects of amph-1 mutants. Amphiphysins thus play a central role in linking the nuclear envelope with the actin and microtubule cytoskeletons. We propose that BIN1 has a direct and evolutionarily conserved role in nuclear positioning, altered in myopathies.

N-WASP is required for Amphiphysin-2/BIN1-dependent nuclear positioning and triad organization in skeletal muscle and is involved in the pathophysiology of centronuclear myopathy.

Mutations in amphiphysin-2/BIN1, dynamin 2, and myotubularin are associated with centronuclear myopathy (CNM), a muscle disorder characterized by myofibers with atypical central nuclear positioning and abnormal triads. Mis-splicing of amphiphysin-2/BIN1 is also associated with myotonic dystrophy that shares histopathological hallmarks with CNM. How amphiphysin-2 orchestrates nuclear positioning and triad organization and how CNM-associated mutations lead to muscle dysfunction remains elusive. We find that N-WASP interacts with amphiphysin-2 in myofibers and that this interaction and N-WASP distribution are disrupted by amphiphysin-2 CNM mutations. We establish that N-WASP functions downstream of amphiphysin-2 to drive peripheral nuclear positioning and triad organization during myofiber formation. Peripheral nuclear positioning requires microtubule/Map7/Kif5b-dependent distribution of nuclei along the myofiber and is driven by actin and nesprins. In adult myofibers, N-WASP and amphiphysin-2 are only involved in the maintenance of triad organization but not in the maintenance of peripheral nuclear positioning. Importantly, we confirmed that N-WASP distribution is disrupted in CNM and myotonic dystrophy patients. Our results support a role for N-WASP in amphiphysin-2-dependent nuclear positioning and triad organization and in CNM and myotonic dystrophy pathophysiology.

Nuclear movement during myotube formation is microtubule and dynein dependent and is regulated by Cdc42, Par6 and Par3.

Cells actively position their nucleus within the cytoplasm. One striking example is observed during skeletal myogenesis. Differentiated myoblasts fuse to form a multinucleated myotube with nuclei positioned in the centre of the syncytium by an unknown mechanism. Here, we describe that the nucleus of a myoblast moves rapidly after fusion towards the central myotube nuclei. This movement is driven by microtubules and dynein/dynactin complex, and requires Cdc42, Par6 and Par3. We found that Par6ß and dynactin accumulate at the nuclear envelope of differentiated myoblasts and myotubes, and this accumulation is dependent on Par6 and Par3 proteins but not on microtubules. These results suggest a mechanism where nuclear movement after fusion is driven by microtubules that emanate from one nucleus that are pulled by dynein/dynactin complex anchored to the nuclear envelope of another nucleus.

Microtubule regulation in mitosis: tubulin phosphorylation by the cyclin-dependent kinase Cdk1.

The activation of the cyclin-dependent kinase Cdk1 at the transition from interphase to mitosis induces important changes in microtubule dynamics. Cdk1 phosphorylates a number of microtubule- or tubulin-binding proteins but, hitherto, tubulin itself has not been detected as a Cdk1 substrate. Here we show that Cdk1 phosphorylates beta-tubulin both in vitro and in vivo. Phosphorylation occurs on Ser172 of beta-tubulin, a site that is well conserved in evolution. Using a phosphopeptide antibody, we find that a fraction of the cell tubulin is phosphorylated during mitosis, and this tubulin phosphorylation is inhibited by the Cdk1 inhibitor roscovitine. In mitotic cells, phosphorylated tubulin is excluded from microtubules, being present in the soluble tubulin fraction. Consistent with this distribution in cells, the incorporation of Cdk1-phosphorylated tubulin into growing microtubules is impaired in vitro. Additionally, EGFP-beta3-tubulin(S172D/E) mutants that mimic phosphorylated tubulin are unable to incorporate into microtubules when expressed in cells. Modeling shows that the presence of a phosphoserine at position 172 may impair both GTP binding to beta-tubulin and interactions between tubulin dimers. These data indicate that phosphorylation of tubulin by Cdk1 could be involved in the regulation of microtubule dynamics during mitosis.