%0 Journal Article
%~ PubMed
%A Morsch, Marco
%A Reddel, Stephen W
%A Ghazanfari, Nazanin
%A Toyka, Klaus V
%A Phillips, William D
%T Muscle Specific Kinase autoantibodies cause synaptic failure through progressive wastage of postsynaptic acetylcholine receptors.
%B Experimental Neurology
%D 2012
%C United States
%I Academic Press
%V 237
%N 2
%P 286-295
%@ 1090-2430
%X In myasthenia gravis muscle weakness is caused by autoantibodies against components of the neuromuscular junction. Patient autoantibodies against Muscle Specific Kinase (MuSK) deplete MuSK from the postsynaptic membrane and reproduce signs of myasthenia gravis when injected into mice. Here we have examined the time-course of structural and functional changes that lead up to synaptic failure. C57Bl6J mice received daily injections of anti-MuSK patient IgG for 15days. Mice began to lose weight from day 12 and demonstrated whole-body weakness by day 14. Electromyography indicated synaptic impairment from day 6 in the gastrocnemius muscle and from day 10 in the diaphragm muscle. Confocal microscopy revealed linear declines in the area and density of postsynaptic acetylcholine receptors (3 - 5% per day) from day 1 through day 15 of the injection series in all five muscles examined. Intracellular recordings from the diaphragm muscle revealed comparable progressive declines in the amplitude of the endplate potential and miniature endplate potential of 3-4% per day. Neither quantal content nor the postsynaptic action potential threshold changed significantly over the injection series. The inverse relationship between the quantal amplitude of a synapse and its quantal content disappeared only late in the injection series (day 10). Our results suggest that the primary myasthenogenic action of anti-MuSK IgG is to cause wastage of postsynaptic acetylcholine receptor density. Consequent reductions in endplate potential amplitudes culminated in failure of neuromuscular transmission.
%Z FOR Codes: 110904


%0 Journal Article
%~ PubMed
%A Ngo, Shyuan T
%A Cole, Rebecca N
%A Sunn, Nana
%A Phillips, William D
%A Noakes, Peter G
%T Neuregulin-1 potentiates agrin-induced acetylcholine receptor clustering through muscle-specific kinase phosphorylation.
%B Journal of Cell Science
%D 2012
%C United Kingdom
%I The Company of Biologists Ltd.
%V 125
%N Pt 6
%P 1531-1543
%@ 1477-9137
%X At neuromuscular synapses, neural agrin (n-agrin) stabilizes embryonic postsynaptic acetylcholine receptor (AChR) clusters by signalling through the muscle-specific kinase (MuSK) complex. Live imaging of cultured myotubes showed that the formation and disassembly of primitive AChR clusters is a dynamic and reversible process favoured by n-agrin, and possibly other synaptic signals. Neuregulin-1 is a growth factor that can act through muscle ErbB receptor kinases to enhance synaptic gene transcription. Recent studies suggest that neuregulin-1-ErbB signalling can modulate n-agrin-induced AChR clustering independently of its effects on transcription. Here we report that neuregulin-1 increased the size of developing AChR clusters when injected into muscles of embryonic mice. We investigated this phenomenon using cultured myotubes, and found that in the ongoing presence of n-agrin, neuregulin-1 potentiates AChR clustering by increasing the tyrosine phosphorylation of MuSK. This potentiation could be blocked by inhibiting Shp2, a postsynaptic tyrosine phosphatase known to modulate the activity of MuSK. Our results provide new evidence that neuregulin-1 modulates the signaling activity of MuSK and hence might function as a second-order regulator of postsynaptic AChR clustering at the neuromuscular synapse. Thus two classic synaptic signalling systems (neuregulin-1 and n-agrin) converge upon MuSK to regulate postsynaptic differentiation.
%Z FOR Codes: 1109 1116


%0 Journal Article
%~ PubMed
%A Gervásio, Othon L
%A Phillips, William D
%A Cole, Louise
%A Allen, David G
%T Caveolae respond to cell stretch and contribute to
stretch-induced signaling.
%B Journal of Cell Science
%D 2011
%C United Kingdom
%I The Company of Biologists Ltd.
%V 124
%N Pt 21
%P 3581-3590
%@ 1477-9137
%X Caveolae are invaginations of the plasma membrane that are formed by caveolins. Caveolar membranes are also enriched in cholesterol, glycosphingolipids and signaling enzymes such as Src kinase. Here we investigate the effect of cell stretch upon caveolar dynamics and signaling. Transfection of C2 myoblasts with caveolin-3-YFP led to the formation of caveolae-like membrane pits 50-100 nm in diameter. Glycosphingolipids became immobilized and tightly packed together within caveolin-rich regions of the plasma membrane. Fluorescence resonance energy transfer (FRET) was used to assess the degree of glycosphingolipid packing. Myoblasts were subjected to a brief (1 minute) stretch on an elastic substratum. Stretch caused a reduction in glycosphingolipid FRET, consistent with a reversible unfolding of caveolar pits in response to membrane tension. Cells expressing caveolin-3-YFP also displayed an enhanced stretch-induced activation of Src kinase, as assessed by immunofluorescence. Repeated stretches resulted in the trafficking and remodeling of caveolin-3-rich membrane domains and accelerated turnover of membrane glycosphingolipids. The stretch-induced unfolding of caveolae, activation of Src and redistribution of caveolin and glycosphingolipids might reflect mechanisms of the cellular adaptation to mechanical stresses.
%Z FOR Codes: 111601


%0 Journal Article
%~ PubMed
%A Ghazanfari, Nazanin
%A Fernandez, Kristine J
%A Murata, Yui
%A Morsch, Marco
%A Ngo, Shyuan T
%A Reddel, Stephen W
%A Noakes, Peter G
%A Phillips, William D
%T Muscle Specific Kinase: organiser of synaptic membrane domains.
%B The international journal of biochemistry & cell biology
%D 2011
%C United Kingdom
%I Pergamon
%V 43
%N 3
%P 295-8
%@ 1357-2725
%X Muscle Specific Kinase (MuSK) is a transmembrane tyrosine kinase vital for forming and maintaining the mammalian neuromuscular junction (NMJ: the synapse between motor nerve and skeletal muscle). MuSK expression switches on during skeletal muscle differentiation. MuSK then becomes restricted to the postsynaptic membrane of the NMJ, where it functions to cluster acetylcholine receptors (AChRs). The expression, activation and turnover of MuSK are each regulated by signals from the motor nerve terminal. MuSK forms the core of an emerging signalling complex that can be acutely activated by neural agrin (N-agrin), a heparin sulfate proteoglycan secreted from the nerve terminal. MuSK activation initiates complex intracellular signalling events that coordinate the local synthesis and assembly of synaptic proteins. The importance of MuSK as a synapse organiser is highlighted by cases of autoimmune myasthenia gravis in which MuSK autoantibodies can deplete MuSK from the postsynaptic membrane, leading to complete disassembly of the adult NMJ.
%Z FOR Codes: 110902 111601


%0 Journal Article
%~ PubMed
%A Cole, Rebecca N
%A Ghazanfari, Nazanin
%A Ngo, Shyuan T
%A Gervasio, Othon L
%A Reddel, Stephen W
%A Phillips, William D
%T Patient autoantibodies deplete postsynaptic Muscle Specific Kinase leading to disassembly of the ACh receptor scaffold and myasthenia gravis in mice.
%B The Journal of physiology
%D 2010
%C United Kingdom
%I Wiley-Blackwell Publishing Ltd.
%V 588
%N 17
%P 3217-29
%@ 1469-7793
%X The postsynaptic muscle-specific kinase (MuSK) coordinates formation of the neuromuscular junction (NMJ) during embryonic development. Here we have studied the effects of MuSK autoantibodies upon the NMJ in adult mice. Daily injections of IgG from four MuSK autoantibody-positive myasthenia gravis patients (MuSK IgG; 45 mg day(1)i.p. for 14 days) caused reductions in postsynaptic ACh receptor (AChR) packing as assessed by fluorescence resonance energy transfer (FRET). IgG from the patients with the highest titres of MuSK autoantibodies caused large (51-73%) reductions in postsynaptic MuSK staining (cf. control mice; P < 0.01) and muscle weakness. Among mice injected for 14 days with control and MuSK patient IgGs, the residual level of MuSK correlated with the degree of impairment of postsynaptic AChR packing. However, the loss of postsynaptic MuSK preceded this impairment of postsynaptic AChR. When added to cultured C2 muscle cells the MuSK autoantibodies caused tyrosine phosphorylation of MuSK and the AChR beta-subunit, and internalization of MuSK from the plasma membrane. The results suggest a pathogenic mechanism in which MuSK autoantibodies rapidly deplete MuSK from the postsynaptic membrane leading to progressive dispersal of postsynaptic AChRs. Moreover, maintenance of postsynaptic AChR packing at the adult NMJ would appear to depend upon physical engagement of MuSK with the AChR scaffold, notwithstanding activation of the MuSK-rapsyn system of AChR clustering.
%Z FOR Codes: 1109 606


%0 Journal Article
%~ PubMed
%A Cole, Rebecca N
%A Reddel, Stephen W
%A Gervásio, Othon L
%A Phillips, William D
%T Anti-MuSK patient antibodies disrupt the mouse neuromuscular junction.
%B Annals of neurology
%D 2008
%C United States
%I John Wiley & Sons, Inc.
%V 63
%N 6
%P 782-9
%@ 1531-8249
%X A subset of myasthenia gravis patients that are seronegative for anti-acetylcholine receptor (anti-AChR) antibodies are instead seropositive for antibodies against the muscle-specific kinase (anti-MuSK-positive). Here, we test whether transfer of IgG from anti-MuSK-positive patients to mice confers impairment of the neuromuscular junction and muscle weakness.
%Z FOR Codes: 1109


%0 Journal Article
%~ PubMed
%A Brockhausen, Jennifer
%A Cole, Rebecca N
%A Gervásio, Othon L
%A Ngo, Shyuan T
%A Noakes, Peter G
%A Phillips, William D
%T Neural agrin increases postsynaptic ACh receptor packing by elevating rapsyn protein at the mouse neuromuscular synapse.
%B Developmental neurobiology
%D 2008
%C United States
%I John Wiley & Sons, Inc.
%V 68
%N 9
%P 1153-69
%@ 1932-8451
%X Fluorescence resonance energy transfer (FRET) experiments at neuromuscular junctions in the mouse tibialis anterior muscle show that postsynaptic acetylcholine receptors (AChRs) become more tightly packed during the first month of postnatal development. Here, we report that the packing of AChRs into postsynaptic aggregates was reduced in 4-week postnatal mice that had reduced amounts of the AChR-associated protein, rapsyn, in the postsynaptic membrane (rapsyn(+/-) mice). We hypothesize that nerve-derived agrin increases postsynaptic expression and targeting of rapsyn, which then drives the developmental increase in AChR packing. Neural agrin treatment elevated the expression of rapsyn in C2 myotubes by a mechanism that involved slowing of rapsyn protein degradation. Similarly, exposure of synapses in postnatal muscle to exogenous agrin increased rapsyn protein levels and elevated the intensity of anti-rapsyn immunofluorescence, relative to AChR, in the postsynaptic membrane. This increase in the rapsyn-to-AChR immunofluorescence ratio was associated with tighter postsynaptic AChR packing and slowed AChR turnover. Acute blockade of synaptic AChRs with alpha-bungarotoxin lowered the rapsyn-to-AChR immunofluorescence ratio, suggesting that AChR signaling also helps regulate the assembly of extra rapsyn in the postsynaptic membrane. The results suggest that at the postnatal neuromuscular synapse agrin signaling elevates the expression and targeting of rapsyn to the postsynaptic membrane, thereby packing more AChRs into stable, functionally-important AChR aggregates.
%Z FOR Codes: 110904


%0 Journal Article
%~ PubMed
%A Gervásio, Othon L
%A Whitehead, Nicholas P
%A Yeung, Ella W
%A Phillips, William D
%A Allen, David G
%T TRPC1 binds to caveolin-3 and is regulated by Src kinase - role in Duchenne muscular dystrophy.
%B Journal of Cell Science
%D 2008
%C United Kingdom
%I Company of Biologists
%V 121
%N 13
%P 2246-2255
%@ 0021-9533
%X Transient receptor potential canonical 1 (TRPC1), a widely expressed calcium (Ca(2+))-permeable channel, is potentially involved in the pathogenesis of Duchenne muscular dystrophy (DMD). Ca(2+) influx through stretch-activated channels, possibly formed by TRPC1, induces muscle-cell damage in the mdx mouse, an animal model of DMD. In this study, we showed that TRPC1, caveolin-3 and Src-kinase protein levels are increased in mdx muscle compared with wild type. TRPC1 and caveolin-3 colocalised and co-immunoprecipitated. Direct binding of TRPC1-CFP to caveolin-3-YFP was confirmed in C2 myoblasts by fluorescence energy resonance transfer (FRET). Caveolin-3-YFP targeted TRPC1-CFP to the plasma membrane. Hydrogen peroxide, a reactive oxygen species (ROS), increased Src activity and enhanced Ca(2+) influx, but only in C2 myoblasts co-expressing TRPC1 and caveolin-3. In mdx muscle, Tiron, a ROS scavenger, and PP2, a Src inhibitor, reduced stretch-induced Ca(2+) entry and increased force recovery. Because ROS production is increased in mdx/DMD, these results suggest that a ROS-Src-TRPC1/caveolin-3 pathway contributes to the pathogenesis of mdx/DMD.
%Z FOR Codes: 110904


%0 Journal Article
%~ PubMed
%A Gervásio, Othon L
%A Armson, Paul F
%A Phillips, William D
%T Developmental increase in the amount of rapsyn per acetylcholine receptor promotes postsynaptic receptor packing and stability.
%B Developmental biology
%D 2007
%C United States
%I Academic Press
%V 305
%N 1
%P 262-275
%@ 0012-1606
%X Neuromuscular synaptic transmission depends upon tight packing of acetylcholine receptors (AChRs) into postsynaptic AChR aggregates, but not all postsynaptic AChRs are aggregated. Here we describe a new confocal Fluorescence Resonance Energy Transfer (FRET) assay for semi-quantitative comparison of the degree to which AChRs are aggregated at synapses. During the first month of postnatal life the mouse tibialis anterior muscle showed increases both in the number of postsynaptic AChRs and the efficiency with which AChR was aggregated (by FRET). There was a concurrent two-fold increase in immunofluorescent labeling for the AChR-associated cytoplasmic protein, rapsyn. When 1-month old muscle was denervated, postsynaptic rapsyn immunostaining was reduced, as was the efficiency of AChR aggregation. In vivo electroporation of rapsyn-EGFP into muscle fibers increased postsynaptic rapsyn levels. Those synapses with higher ratios of rapsyn-EGFP to AChR displayed a slower metabolic turnover of AChR. Conversely, the reduction of postsynaptic rapsyn after denervation was accompanied by an acceleration of AChR turnover. Thus, a developmental increase in the amount of rapsyn targeted to the postsynaptic membrane may drive enhanced postsynaptic AChRs aggregation and AChR stability within the postsynaptic membrane.
%Z FOR Codes: 110902 110905


%0 Journal Article
%~ PubMed
%A Ngo, Shyuan T
%A Noakes, Peter G
%A Phillips, William D
%T Neural agrin: a synaptic stabiliser.
%B The international journal of biochemistry & cell biology
%D 2007
%C United Kingdom
%I Pergamon
%V 39
%N 5
%P 863-867
%@ 1357-2725
%X Neural agrin is a heparan sulphate proteoglycan first defined by its ability to induce the clustering of acetylcholine receptors (AChRs) on cultured muscle cells. Neural agrin activates the transmembrane Muscle Specific Kinase (MuSK) on the postsynaptic muscle cell to stabilise the developing neuromuscular synapse. Three biological mechanisms for agrin/MuSK signalling are briefly discussed: selective transcription of synaptic genes such as MuSK itself, to reinforce developing postsynaptic clusters of AChRs; initiation of second messenger signalling pathways that can induce the formation of AChR clusters and retrograde signalling downstream of agrin/MuSK that may transform the growth cone of the motor axon into a stable differentiated nerve terminal, specialised for regulated exocytosis of neurotransmitter. Here we briefly review some key mechanisms through which neural agrin acts to foster the formation of mature neuromuscular synapses.
%Z FOR Codes: 110902 110905