- Email: [email protected]
Investigating the role of nidogens, basement membrane proteins, at theneuromuscular junction in health and disease
S34
Abstracts of the 10th Neuromuscular Translational Research Conference / Neuromuscular Disorder 27S1 (2017) S5–S44
NMJ+C03 Characterisation of MYO9A as a pre-synaptic CMS gene E. O’Connor, I. Cordts, A. Roos, H. Lochmüller John Walton Muscular Dystrophy Research Centre, Newcastle University Institute of Genetic Medicine, Newcastle-upon-Tyne, UK E-mail: [email protected] Background: Congenital myasthenic syndromes (CMS) are a group of disorders characterised by compromised function of the NMJ. CMS usually manifests in childhood with fatigable weakness of limb, ocular and bulbar muscles depending on the underlying genetic defect. Advances in research for neuromuscular disorders, including the generation of next generation sequencing techniques, has accelerated the identification of CMS-related genes. However, for many subtypes, the precise pathomechanism has not yet been characterised. Aims: To unravel the pathophysiology underlying CMS caused by mutations in MYO9A. We hypothesised that defects in MYO9A affect the neuronal cytoskeleton, and thus lead to impaired vesicular transport. Methods: NSC-34 cells (mouse motor neuron-derived cell-line), depleted for MYO9A, were used to assess the effect of reduced MYO9A expression on the cytoskeleton using immunofluorescent and immunoblotting techniques. Vesicular transport was analysed using three main assays; a secretome study to observe effects on secreted proteins from NSC-34 cells, a transport assay to assess the transport of newly synthesised proteins to the cell surface and a recycling assay to look at receptor recycling processes. In addition, an unbiased approach utilising label-free comparative proteomic profiling of wild-type and MYO9A-depleted NSC-34 cells was performed to identify key players of the pathophysiology. Results: Disruption of the cytoskeleton has been identified in cells depleted for MYO9A, with an upregulation of actin and a downregulation of other structural proteins. Accordingly, defects in receptor recycling and regular transport of proteins to the cell surface were also observed in cells depleted for MYO9A. Proteomic data support a role for defective vesicular transport in NSC-34 cells and identified affected proteins which are also involved in the manifestation of further neuromuscular disorders. Conclusions: Our combined data allow new insights into the pathophysiology of CMS and show that loss of MYO9A affects the neuronal cytoskeleton, and leads to impaired transport and vesicular recycling of proteins. This could lead to a CMS phenotype by affecting the surface expression and secretion of important NMJ proteins, as well as the structure of the nerve terminal. NMJ+C04 Investigating the role of nidogens, basement membrane proteins, at the neuromuscular junction in health and disease I.F.G. Meyer, G. Schiavo Sobell Dept. Institute of Neurology, University College London, Queen Square, London, UK E-mail: [email protected] Background: A number of neuromuscular diseases such as ALS, CMT and SMA are characterised by progressive muscle wasting and neuronal degeneration. Alterations to the neuromuscular junction (NMJ) are one of the earliest pathological events in these diseases and have been shown to precede motor neuron cell death and clinical symptoms. This suggests that the muscle may play an active role in neuronal death, contributing to the pathology or perhaps even initiating it. Interestingly, some muscle types appear to be more vulnerable to disease, with fast-twitch fibres and their innervating neurons degenerating first. Together, these findings suggest the presence of muscle-associated factors that influence the susceptibility of certain muscle fibre types to degeneration. It is therefore important to investigate the homeostatic mechanisms of the NMJ to better understand these pathological events. Previous work in our lab has identified an endocytic trafficking pathway at the neuromuscular junction, involving the release of an extracellular protein, nidogen, from the synaptic basement membrane and its subsequent internalisation into motor neurons. We aim to investigate the endogenous interactions and intracellular functions of this protein using neuronal cultures and ex vivo murine muscle samples with a combination of molecular, biochemical and imaging techniques. Clarification of this trafficking route may lead to identification of a novel signalling pathway at the neuromuscular junction, which would further our understanding of the
development and maintenance of this extremely important type of synapse. Molecular components of this pathway are likely to provide new therapeutic targets for the treatment of neuromuscular diseases. NMJ+C05 Improving genetic diagnosis and counselling for patients with myotonia congenita K. Suetterlin1, R. Sud2, J. Burge1, S. McCall2, D. Fialho1, A. Haworth2, M.G. Sweeney2, H. Houlden1,2, S. Schorge3, E. Matthews1, M.G. Hanna1, R. Männikkö1 1 MRC Centre for Neuromuscular Diseases, Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK; 2Neurogenetics Department, National Hospital for Neurology and Neurosurgery, Queen Square, London, UK; 3Department of Clinical and Experimental Epilepsy, UCL Institute of Neurology, London, UK Background: Myotonia congenita is caused by mutations in the muscle chloride channel gene (CLCN1). Accuracy of diagnosis and genetic counselling can be challenging for missense mutations which have been reported as dominant or recessive, are found throughout the length of the channel and have no clear structure/function relationship. Aim: To improve the accuracy of diagnosis and genetic counselling for patients with myotonia congenita Methods or Patients or Materials: ClC-1 missense variants identified in patients referred to our national diagnostic centre were functionally characterised and mapped onto a model of ClC-1. As segregation data was not always complete, probands were categorised according to the reported inheritance pattern of myotonic symptoms. Results: 211 probands with 80 different CLCN1 missense variants were included. Variants outside of the first half of the channel transmembrane region (111-347) were significantly less likely to be associated with a dominant inheritance pattern (p=0.00006). Variants in the cytoplasmic regions were significantly more likely to be categorised as uncertain pathogenicity (p=0.004). Virtually all variants (98%) could be categorised as one of 4 distinct functional groups (Voltage Shift 1 and 2, Functionally Recessive and Wild-type like). By combining variant location and functional group the risk of associated inheritance pattern could be stratified e.g. 44% of all variants in residues 111–347 were associated with a dominant inheritance pattern, this increased to 94.5% if limited to voltage shift 1 variants and reduced to 0% if limited to functionally Wild-type like variants. Only 5.3% of variants in the second half of the transmembrane region were associated with a dominant inheritance pattern. All of these variants were voltage shift variants. In the cytoplasmic region 58% of the functionally wild-type like variants were categorised as of uncertain pathogenicity versus 0% of voltage shifting or functionally recessive variants. Conclusions: Combining clinical, functional and genetic data with molecular modelling has enabled us to develop a framework for interpreting the clinical significance of ClC-1 variants. This will contribute significantly to diagnosis and genetic counselling in myotonia congenita. NMJ+C06 Recessive SCN4A loss of function in congenital myasthenic syndrome, congenital myopathy or fetal akinesia deformation sequence M.G. Thor1,2, M.G. Hanna1,2, F. Muntoni1,2,3, R. Männikkö1,2 1 MRC Centre for Neuromuscular Diseases, Queen Square, London, UK; 2 Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK; 3Dubowitz Neuromuscular Centre, UCL Institute of Child Health, London, UK E-mail: [email protected] Background: SCN4A encodes NaV1.4, the skeletal muscle sodium channel integral to action potential generation and propagation. While dominant gain of function mutations are a well-established cause of myotonia and periodic paralysis, the phenotypes associated with recessive loss of function are more diffuse. Loss of function has been associated with a few cases of congenital myasthenic syndrome. We further explore recessive SCN4A mutations in disease. Methods: Using whole-exome sequencing, homozygous or compound heterozygous SCN4A mutations were identified in twelve families. NaV1.4 channel variants were expressed in Xenopus laevis oocytes and HEK293 cells,
Abstracts of the 10th Neuromuscular Translational Research Conference / Neuromuscular Disorder 27S1 (2017) S5–S44
NMJ+C03 Characterisation of MYO9A as a pre-synaptic CMS gene E. O’Connor, I. Cordts, A. Roos, H. Lochmüller John Walton Muscular Dystrophy Research Centre, Newcastle University Institute of Genetic Medicine, Newcastle-upon-Tyne, UK E-mail: [email protected] Background: Congenital myasthenic syndromes (CMS) are a group of disorders characterised by compromised function of the NMJ. CMS usually manifests in childhood with fatigable weakness of limb, ocular and bulbar muscles depending on the underlying genetic defect. Advances in research for neuromuscular disorders, including the generation of next generation sequencing techniques, has accelerated the identification of CMS-related genes. However, for many subtypes, the precise pathomechanism has not yet been characterised. Aims: To unravel the pathophysiology underlying CMS caused by mutations in MYO9A. We hypothesised that defects in MYO9A affect the neuronal cytoskeleton, and thus lead to impaired vesicular transport. Methods: NSC-34 cells (mouse motor neuron-derived cell-line), depleted for MYO9A, were used to assess the effect of reduced MYO9A expression on the cytoskeleton using immunofluorescent and immunoblotting techniques. Vesicular transport was analysed using three main assays; a secretome study to observe effects on secreted proteins from NSC-34 cells, a transport assay to assess the transport of newly synthesised proteins to the cell surface and a recycling assay to look at receptor recycling processes. In addition, an unbiased approach utilising label-free comparative proteomic profiling of wild-type and MYO9A-depleted NSC-34 cells was performed to identify key players of the pathophysiology. Results: Disruption of the cytoskeleton has been identified in cells depleted for MYO9A, with an upregulation of actin and a downregulation of other structural proteins. Accordingly, defects in receptor recycling and regular transport of proteins to the cell surface were also observed in cells depleted for MYO9A. Proteomic data support a role for defective vesicular transport in NSC-34 cells and identified affected proteins which are also involved in the manifestation of further neuromuscular disorders. Conclusions: Our combined data allow new insights into the pathophysiology of CMS and show that loss of MYO9A affects the neuronal cytoskeleton, and leads to impaired transport and vesicular recycling of proteins. This could lead to a CMS phenotype by affecting the surface expression and secretion of important NMJ proteins, as well as the structure of the nerve terminal. NMJ+C04 Investigating the role of nidogens, basement membrane proteins, at the neuromuscular junction in health and disease I.F.G. Meyer, G. Schiavo Sobell Dept. Institute of Neurology, University College London, Queen Square, London, UK E-mail: [email protected] Background: A number of neuromuscular diseases such as ALS, CMT and SMA are characterised by progressive muscle wasting and neuronal degeneration. Alterations to the neuromuscular junction (NMJ) are one of the earliest pathological events in these diseases and have been shown to precede motor neuron cell death and clinical symptoms. This suggests that the muscle may play an active role in neuronal death, contributing to the pathology or perhaps even initiating it. Interestingly, some muscle types appear to be more vulnerable to disease, with fast-twitch fibres and their innervating neurons degenerating first. Together, these findings suggest the presence of muscle-associated factors that influence the susceptibility of certain muscle fibre types to degeneration. It is therefore important to investigate the homeostatic mechanisms of the NMJ to better understand these pathological events. Previous work in our lab has identified an endocytic trafficking pathway at the neuromuscular junction, involving the release of an extracellular protein, nidogen, from the synaptic basement membrane and its subsequent internalisation into motor neurons. We aim to investigate the endogenous interactions and intracellular functions of this protein using neuronal cultures and ex vivo murine muscle samples with a combination of molecular, biochemical and imaging techniques. Clarification of this trafficking route may lead to identification of a novel signalling pathway at the neuromuscular junction, which would further our understanding of the
development and maintenance of this extremely important type of synapse. Molecular components of this pathway are likely to provide new therapeutic targets for the treatment of neuromuscular diseases. NMJ+C05 Improving genetic diagnosis and counselling for patients with myotonia congenita K. Suetterlin1, R. Sud2, J. Burge1, S. McCall2, D. Fialho1, A. Haworth2, M.G. Sweeney2, H. Houlden1,2, S. Schorge3, E. Matthews1, M.G. Hanna1, R. Männikkö1 1 MRC Centre for Neuromuscular Diseases, Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK; 2Neurogenetics Department, National Hospital for Neurology and Neurosurgery, Queen Square, London, UK; 3Department of Clinical and Experimental Epilepsy, UCL Institute of Neurology, London, UK Background: Myotonia congenita is caused by mutations in the muscle chloride channel gene (CLCN1). Accuracy of diagnosis and genetic counselling can be challenging for missense mutations which have been reported as dominant or recessive, are found throughout the length of the channel and have no clear structure/function relationship. Aim: To improve the accuracy of diagnosis and genetic counselling for patients with myotonia congenita Methods or Patients or Materials: ClC-1 missense variants identified in patients referred to our national diagnostic centre were functionally characterised and mapped onto a model of ClC-1. As segregation data was not always complete, probands were categorised according to the reported inheritance pattern of myotonic symptoms. Results: 211 probands with 80 different CLCN1 missense variants were included. Variants outside of the first half of the channel transmembrane region (111-347) were significantly less likely to be associated with a dominant inheritance pattern (p=0.00006). Variants in the cytoplasmic regions were significantly more likely to be categorised as uncertain pathogenicity (p=0.004). Virtually all variants (98%) could be categorised as one of 4 distinct functional groups (Voltage Shift 1 and 2, Functionally Recessive and Wild-type like). By combining variant location and functional group the risk of associated inheritance pattern could be stratified e.g. 44% of all variants in residues 111–347 were associated with a dominant inheritance pattern, this increased to 94.5% if limited to voltage shift 1 variants and reduced to 0% if limited to functionally Wild-type like variants. Only 5.3% of variants in the second half of the transmembrane region were associated with a dominant inheritance pattern. All of these variants were voltage shift variants. In the cytoplasmic region 58% of the functionally wild-type like variants were categorised as of uncertain pathogenicity versus 0% of voltage shifting or functionally recessive variants. Conclusions: Combining clinical, functional and genetic data with molecular modelling has enabled us to develop a framework for interpreting the clinical significance of ClC-1 variants. This will contribute significantly to diagnosis and genetic counselling in myotonia congenita. NMJ+C06 Recessive SCN4A loss of function in congenital myasthenic syndrome, congenital myopathy or fetal akinesia deformation sequence M.G. Thor1,2, M.G. Hanna1,2, F. Muntoni1,2,3, R. Männikkö1,2 1 MRC Centre for Neuromuscular Diseases, Queen Square, London, UK; 2 Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK; 3Dubowitz Neuromuscular Centre, UCL Institute of Child Health, London, UK E-mail: [email protected] Background: SCN4A encodes NaV1.4, the skeletal muscle sodium channel integral to action potential generation and propagation. While dominant gain of function mutations are a well-established cause of myotonia and periodic paralysis, the phenotypes associated with recessive loss of function are more diffuse. Loss of function has been associated with a few cases of congenital myasthenic syndrome. We further explore recessive SCN4A mutations in disease. Methods: Using whole-exome sequencing, homozygous or compound heterozygous SCN4A mutations were identified in twelve families. NaV1.4 channel variants were expressed in Xenopus laevis oocytes and HEK293 cells,