Prof David Beeson

Research Area: Ion Channels and Disease
Scientific Themes: Molecular, Cell & Systems Biology and Genes, Genetics, Epigenetics & Genomics
Keywords: Ion channels, AChR, Congenital myasthenic syndromes, neuromuscular junction, synaptic disorders
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Fluorescence images of the neuromuscular junction stained with antibodies to the acetylcholine receptor (red) and terminal region of the motor nerve (green)

Fluorescence images of the neuromuscular junction stained with antibodies to the acetylcholine recep ...

Fluorescence images of the neuromuscular junction stained with antibodies to the acetylcholine receptor (red) and terminal region of the motor nerve (green)

Fluorescence images of the neuromuscular junction stained with antibodies to the acetylcholine recep ...

Molecular Neurosciences forms an integral part of the Neurosciences Group, and collaborates with Professor Vincent, Professor Willcox and Dr Lang.

We are studying inherited diseases that affect neuromuscular transmission, focusing both on mutations that directly affect the muscle acetylcholine receptors (AChR) and on mutations in proteins that control synaptic structure and formation. The neuromuscular junction is both well understood and accessible for study. Functional analysis of mutations at the molecular level can be directly correlated with measurements of defective synaptic transmission in vivo and with the clinical features of the patients. Thus a detailed knowledge of inherited dysfunction of neuromuscular transmission forms a paradigm for investigation of other neurological syndromes that may result from defective synaptic transmission in the CNS.

At the clinical level the congenital myasthenic syndromes provide a fascinating series of subtly different phenotypes that often require different treatments. Although a series of different syndromes have been characterized in detail, including the slow channel syndrome, fast channel syndrome, AChR deficiency syndrome acetylcholinesterase deficiency, and CMS due to DOK7 mutations, in about 40% of cases the underlying genetics is still unknown.

Our present work aims to identify candidate genes, define underlying mutations, investigate the disease mechanisms at the molecular level, and then explore new therapeutic strategies.

The projects involve close collaboration with clinicians in the Department of Clinical Neurology in Oxford, and with neurologists from the UK and overseas. The success of the work has led to the group being commissioned to provide a National Service for mutation detection and treatment of congenital myasthenic syndromes, as part of a consortium chosen by the National Specialist Advisory Commissioning Service for the diagnosis of rare muscle diseases. The service provides a means for the rapid translation of discoveries in the laboratory into clinical practice.

Our studies also include a series of laboratory-based collaborative projects. Studies on proteins involved in neuromuscular junction synaptogenesis are performed with Professor Yamanashi (Tokyo Medical and Dental School, Japan), and gene therapies in disease models using catalytic nucleic acids is being studied with Dr Matthew Wood, Department of Human Anatomy, Oxford. In addition, we make up part of the Wellcome Trust-funded OXION consortium that aims to understand the function of ion channels and their role in disease.

Name Department Institution Country
Prof Yuji Yamanashi Tokyo Dental Hospital Japan

Zoltowska K, Webster R, Finlayson S, Maxwell S, Cossins J, Müller J, Lochmüller H, Beeson D. 2013. Mutations in GFPT1 that underlie limb-girdle congenital myasthenic syndrome result in reduced cell-surface expression of muscle AChR. Hum Mol Genet, 22 (14), pp. 2905-2913. Read abstract | Read more

Mutations in GFPT1 underlie a congenital myasthenic syndrome (CMS) characterized by a limb-girdle pattern of muscle weakness. Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is a key rate-limiting enzyme in the hexosamine biosynthetic pathway providing building blocks for the glycosylation of proteins and lipids. It is expressed ubiquitously and it is not readily apparent why mutations in this gene should cause a syndrome with symptoms restricted to muscle and, in particular, to the neuromuscular junction. Data from a muscle biopsy obtained from a patient with GFPT1 mutations indicated that there were reduced endplate acetylcholine receptors. We, therefore, further investigated the relationship between identified mutations in GFPT1 and expression of the muscle acetylcholine receptor. Cultured myotubes derived from two patients with GFPT1 mutations showed a significant reduction in cell-surface AChR expression (Pt1 P < 0.0001; Pt2 P = 0.0097). Inhibition of GFPT1 enzymatic activity or siRNA silencing of GFPT1 expression both resulted in reduced AChR cell-surface expression. Western blot and gene-silencing experiments indicate this is due to reduced steady-state levels of AChR α, δ, ε, but not β subunits rather than altered transcription of AChR-subunit RNA. Uridine diphospho-N-acetylglucosamine, a product of the hexosamine synthetic pathway, acts as a substrate at an early stage in the N-linked glycosylation pathway. Similarity between CMS due to GFPT1 mutations and CMS due to DPAGT1 mutations would suggest that reduced endplate AChR due to defective N-linked glycosylation is a primary disease mechanism in this disorder. Hide abstract

Finlayson S, Beeson D, Palace J. 2013. Congenital myasthenic syndromes: an update. Pract Neurol, 13 (2), pp. 80-91. | Read more

Cossins J, Belaya K, Hicks D, Salih MA, Finlayson S, Carboni N, Liu WW, Maxwell S et al. 2013. Congenital myasthenic syndromes due to mutations in ALG2 and ALG14. Brain, 136 (Pt 3), pp. 944-956. Read abstract | Read more

Congenital myasthenic syndromes are a heterogeneous group of inherited disorders that arise from impaired signal transmission at the neuromuscular synapse. They are characterized by fatigable muscle weakness. We performed linkage analysis, whole-exome and whole-genome sequencing to determine the underlying defect in patients with an inherited limb-girdle pattern of myasthenic weakness. We identify ALG14 and ALG2 as novel genes in which mutations cause a congenital myasthenic syndrome. Through analogy with yeast, ALG14 is thought to form a multiglycosyltransferase complex with ALG13 and DPAGT1 that catalyses the first two committed steps of asparagine-linked protein glycosylation. We show that ALG14 is concentrated at the muscle motor endplates and small interfering RNA silencing of ALG14 results in reduced cell-surface expression of muscle acetylcholine receptor expressed in human embryonic kidney 293 cells. ALG2 is an alpha-1,3-mannosyltransferase that also catalyses early steps in the asparagine-linked glycosylation pathway. Mutations were identified in two kinships, with mutation ALG2p.Val68Gly found to severely reduce ALG2 expression both in patient muscle, and in cell cultures. Identification of DPAGT1, ALG14 and ALG2 mutations as a cause of congenital myasthenic syndrome underscores the importance of asparagine-linked protein glycosylation for proper functioning of the neuromuscular junction. These syndromes form part of the wider spectrum of congenital disorders of glycosylation caused by impaired asparagine-linked glycosylation. It is likely that further genes encoding components of this pathway will be associated with congenital myasthenic syndromes or impaired neuromuscular transmission as part of a more severe multisystem disorder. Our findings suggest that treatment with cholinesterase inhibitors may improve muscle function in many of the congenital disorders of glycosylation. Hide abstract

Belaya K, Finlayson S, Slater CR, Cossins J, Liu WW, Maxwell S, McGowan SJ, Maslau S et al. 2012. Mutations in DPAGT1 cause a limb-girdle congenital myasthenic syndrome with tubular aggregates. Am J Hum Genet, 91 (1), pp. 193-201. Read abstract | Read more

Congenital myasthenic syndromes are a heterogeneous group of inherited disorders that arise from impaired signal transmission at the neuromuscular synapse. They are characterized by fatigable muscle weakness. We performed whole-exome sequencing to determine the underlying defect in a group of individuals with an inherited limb-girdle pattern of myasthenic weakness. We identify DPAGT1 as a gene in which mutations cause a congenital myasthenic syndrome. We describe seven different mutations found in five individuals with DPAGT1 mutations. The affected individuals share a number of common clinical features, including involvement of proximal limb muscles, response to treatment with cholinesterase inhibitors and 3,4-diaminopyridine, and the presence of tubular aggregates in muscle biopsies. Analyses of motor endplates from two of the individuals demonstrate a severe reduction of endplate acetylcholine receptors. DPAGT1 is an essential enzyme catalyzing the first committed step of N-linked protein glycosylation. Our findings underscore the importance of N-linked protein glycosylation for proper functioning of the neuromuscular junction. Using the DPAGT1-specific inhibitor tunicamycin, we show that DPAGT1 is required for efficient glycosylation of acetylcholine-receptor subunits and for efficient export of acetylcholine receptors to the cell surface. We suggest that the primary pathogenic mechanism of DPAGT1 mutations is reduced levels of acetylcholine receptors at the endplate region. These individuals share clinical features similar to those of congenital myasthenic syndrome due to GFPT1 mutations, and their disorder might be part of a larger subgroup comprising the congenital myasthenic syndromes that result from defects in the N-linked glycosylation pathway and that manifest through impaired neuromuscular transmission. Hide abstract

Cossins J, Liu WW, Belaya K, Maxwell S, Oldridge M, Lester T, Robb S, Beeson D. 2012. The spectrum of mutations that underlie the neuromuscular junction synaptopathy in DOK7 congenital myasthenic syndrome. Hum Mol Genet, 21 (17), pp. 3765-3775. Read abstract | Read more

Congenital myasthenic syndromes (CMS) are a group of inherited diseases that affect synaptic transmission at the neuromuscular junction and result in fatiguable muscle weakness. A subgroup of CMS patients have a recessively inherited limb-girdle pattern of weakness caused by mutations in DOK7. DOK7 encodes DOK7, an adaptor protein that is expressed in the skeletal muscle and heart and that is essential for the development and maintenance of the neuromuscular junction. We have screened the DOK7 gene for mutations by polymerase chain reaction amplification and bi-directional sequencing of exonic and promoter regions and performed acetylcholine receptor (AChR) clustering assays and used exon trapping to determine the pathogenicity of detected variants. Approximately 18% of genetically diagnosed CMSs in the UK have mutations in DOK7, with mutations in this gene identified in more than 60 kinships to date. Thirty-four different pathogenic mutations were identified as well as 27 variants likely to be non-pathogenic. An exon 7 frameshift duplication c.1124_1127dupTGCC is commonly found in at least one allele. We analyse the effect of the common frameshift c.1124_1127dupTGCC and show that 10/11 suspected missense mutations have a deleterious effect on AChR clustering. We identify for the first time homozygous or compound heterozygous mutations that are localized 5' to exon 7. In addition, three silent variants in the N-terminal half of DOK7 are predicted to alter the splicing of the DOK7 RNA transcript. The DOK7 gene is highly polymorphic, and within these many variants, we define a spectrum of mutations that can underlie DOK7 CMS that will inform in managing this disorder. Hide abstract

Webster R, Maxwell S, Spearman H, Tai K, Beckstein O, Sansom M, Beeson D. 2012. A novel congenital myasthenic syndrome due to decreased acetylcholine receptor ion-channel conductance Brain, 135 (4), pp. 1070-1080. Read abstract | Read more

Muscle acetylcholine receptor ion channels mediate neurotransmission by depolarizing the postsynaptic membrane at the neuromuscular junction. Inherited disorders of neuromuscular transmission, termed congenital myasthenic syndromes, are commonly caused by mutations in genes encoding the five subunits of the acetylcholine receptor that severely reduce endplate acetylcholine receptor numbers and/or cause kinetic abnormalities of acetylcholine receptor function. We tracked the cause of the myasthenic disorder in a female with onset of first symptoms at birth, who displayed mildly progressive bulbar, respiratory and generalized limb weakness with ptosis and ophthalmoplegia. Direct DNA sequencing revealed heteroallelic mutations in exon 8 of the acetylcholine receptor ε-subunit gene. Two alleles were identified: one with the missense substitution p.εP282R, and the second with a deletion, c.798-800delCTT, which result in the loss of a single amino acid, residue F266, within the M2 transmembrane domain. When these acetylcholine receptor mutations were expressed in HEK 293 cells, the p.εP282R mutation caused severely reduced expression on the cell surface, whereas p.εΔF266 gave robust surface expression. Single-channel analysis for p.εΔF266 acetylcholine receptor channels showed the longest burst duration population was not different from wild-type acetylcholine receptor (4.39±0.6ms versus 4.68±0.7ms, n=5 each) but that the amplitude of channel openings was reduced. Channel amplitudes at different holding potentials showed that single-channel conductance was significantly reduced in p.εΔF266 acetylcholine receptor channels (42.7±1.4 pS, n=8, compared with 70.9±1.6 pS for wild-type, n=6). Although a phenylalanine residue at this position within M2 is conserved throughout ligand-gated excitatory cys-loop channel subunits, deletion of equivalent residues in the other subunits of muscle acetylcholine receptor did not have equivalent effects. Modelling the impact of p.εΔF266 revealed only a minor alteration to channel structure. In this study we uncover the novel mechanism of reduced acetylcholine receptor channel conductance as an underlying cause of congenital myasthenic syndrome, with the 'low conductance' phenotype that results from the p.εΔF266 deletion mutation revealed by the coinheritance of the low-expressor mutation p.εP282R. © 2012 The Author. Hide abstract

Lashley D, Palace J, Jayawant S, Robb S, Beeson D. 2010. Ephedrine treatment in congenital myasthenic syndrome due to mutations in DOK7. Neurology, 74 (19), pp. 1517-1523. Read abstract | Read more

BACKGROUND: Mutations in the postsynaptic adaptor protein Dok-7 underlie congenital myasthenic syndrome (CMS) with a characteristic limb girdle pattern of muscle weakness. Patients usually do not respond to or worsen with the standard CMS treatments: cholinesterase inhibitors and 3,4-diaminopyridine. However, anecdotal reports suggest they may improve with ephedrine. METHODS: This was an open prospective follow-up study to determine muscle strength in response to ephedrine in Dok-7 CMS. Patients were first evaluated as inpatients for suitability for a trial of treatment with ephedrine. The response was assessed at 2 and 6 to 8 months follow-up clinic visits using a quantitative myasthenia gravis (severity) score (QMG) and mobility measures. RESULTS: Ten out of 12 of the cohort with DOK7 mutations tolerated ephedrine. We noted a progressive response to treatment over the 6 to 8 months assessment period with a significant improvement at the final QMG score (p = 0.009). Mobility scores also improved (p = 0.0006). Improvements in the subcomponents of the QMG score that measured proximal muscle function (those muscle groups most severely affected) were most marked, and in some cases were dramatic. All patients reported enhanced activities of daily living at 6-8 months. CONCLUSION: Ephedrine appears to be an effective treatment for Dok-7 CMS. It is well-tolerated by most patients and improvement in strength can be profound. Determining the long-term response and the most effective dosing regimen will require further research. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that ephedrine given at doses between 15 and 90 mg/day improves muscle strength in patients with documented mutations in DOK7. Hide abstract

Giraud M, Taubert R, Vandiedonck C, Ke X, Lévi-Strauss M, Pagani F, Baralle FE, Eymard B et al. 2007. An IRF8-binding promoter variant and AIRE control CHRNA1 promiscuous expression in thymus. Nature, 448 (7156), pp. 934-937. Read abstract | Read more

Promiscuous expression of tissue-restricted auto-antigens in the thymus imposes T-cell tolerance and provides protection from autoimmune diseases. Promiscuous expression of a set of self-antigens occurs in medullary thymic epithelial cells and is partly controlled by the autoimmune regulator (AIRE), a nuclear protein for which loss-of-function mutations cause the type 1 autoimmune polyendocrine syndrome. However, additional factors must be involved in the regulation of this promiscuous expression. Here we describe a mechanism controlling thymic transcription of a prototypic tissue-restricted human auto-antigen gene, CHRNA1. This gene encodes the alpha-subunit of the muscle acetylcholine receptor, which is the main target of pathogenic auto-antibodies in autoimmune myasthenia gravis. On re-sequencing the CHRNA1 gene, we identified a functional bi-allelic variant in the promoter that is associated with early onset of disease in two independent human populations (France and United Kingdom). We show that this variant prevents binding of interferon regulatory factor 8 (IRF8) and abrogates CHRNA1 promoter activity in thymic epithelial cells in vitro. Notably, both the CHRNA1 promoter variant and AIRE modulate CHRNA1 messenger RNA levels in human medullary thymic epithelial cells ex vivo and also in a transactivation assay. These findings reveal a critical function of AIRE and the interferon signalling pathway in regulating quantitative expression of this auto-antigen in the thymus, suggesting that together they set the threshold for self-tolerance versus autoimmunity. Hide abstract

Cossins J, Webster R, Maxwell S, Burke G, Vincent A, Beeson D. 2004. A mouse model of AChR deficiency syndrome with a phenotype reflecting the human condition. Hum Mol Genet, 13 (23), pp. 2947-2957. Read abstract | Read more

The two subtypes of mammalian muscle nicotinic acetylcholine receptors (AChR) are generated by the substitution of the epsilon (adult) subunit for the gamma (fetal) subunit within the AChR pentamer. Null mutations of the adult AChR epsilon-subunit gene are the most common cause of the AChR deficiency syndrome. This is a disorder of neuromuscular transmission characterized by non-progressive fatigable muscle weakness present throughout life. In contrast with the human disorder, mice with AChR epsilon-subunit null mutations die between 10 and 14 weeks of age. We generated transgenic mice that constitutively express the human AChR gamma-subunit in an AChR epsilon-subunit 'knock-out' background. These mice, in which neuromuscular transmission is mediated by fetal AChR, live well into adult life but show striking similarities to human AChR deficiency syndrome. They display fatigable muscle weakness, reduced miniature endplate potentials and endplate potentials, reduced motor endplate AChR number and altered endplate morphology. Our results illustrate how species differences in the control of ion-channel gene expression may affect disease phenotype, demonstrate that expression of adult AChR subtype is not essential for long-term survival, and suggest that in patients with AChR deficiency syndrome, up-regulation of the gamma-subunit could be a beneficial therapeutic strategy. Hide abstract

Abdelgany A, Wood M, Beeson D. 2003. Allele-specific silencing of a pathogenic mutant acetylcholine receptor subunit by RNA interference. Hum Mol Genet, 12 (20), pp. 2637-2644. Read abstract | Read more

Slow channel congenital myasthenic syndrome (SCCMS) is a disorder of the neuromuscular synapse caused by dominantly inherited missense mutations in genes that encode the muscle acetylcholine receptor (AChR) subunits. Here we investigate the potential of post-transcriptional gene silencing using RNA interference (RNAi) for the selective down-regulation of pathogenic mutant AChR. By transfection of both siRNA and shRNA into mammalian cells expressing wild-type or mutant AChR subunits, we show, using 125I-alpha-bungarotoxin binding and immunofluorescence to measure cell surface AChR expression, efficient discrimination between the silencing of alphaS226F AChR mutant RNA transcripts and the wild-type. In this model we find that selectivity between mutant and wild-type transcripts is optimized with the nucleotide mismatch at position 9 in the shRNA complementary sequence. We also find that allele-specific silencing using shRNA has comparable efficiency to that using siRNA, underlining the general potential of stable expression of shRNA molecules as a long term therapeutic approach for allele-specific silencing of mutant transcripts in dominant genetic disorders. Hide abstract

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