Publications by authors named "Ruth Herbst"

20 Publications

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MuSk function during health and disease.

Authors:
Ruth Herbst

Neurosci Lett 2020 01 4;716:134676. Epub 2019 Dec 4.

Medical University of Vienna, Center for Pathophysiology, Infectiology and Immunology, Kinderspitalgasse 15, 1090 Vienna, Austria. Electronic address:

The receptor tyrosine kinase MuSK (muscle-specific kinase) is the key signaling molecule during the formation of a mature and functional neuromuscular junction (NMJ). Signal transduction events downstream of MuSK activation induce both pre- and postsynaptic differentiation, which, most prominently, includes the clustering of acetylcholine receptors (AChRs) at synaptic sites. MuSK activation requires a complex interplay between its co-receptor Lrp4 (low-density lipoprotein receptor-related protein-4), the motor neuron-derived heparan-sulfate proteoglycan Agrin and the intracellular adaptor protein Dok-7. A tight regulation of MuSK kinase activity is crucial for proper NMJ development. Defects in MuSK signaling are the cause of muscle weakness as reported in congenital myasthenic syndromes and myasthenia gravis. This review focuses on recent structure-based analyses of MuSK, Agrin, Lrp4 and Dok-7 interactions and their function during MuSK activation. Conclusions about the regulation of the MuSK kinase that were derived from molecular structures will be highlighted. In addition, the role of MuSK during development and disease will be discussed.
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http://dx.doi.org/10.1016/j.neulet.2019.134676DOI Listing
January 2020

Congenital myasthenic syndrome due to mutations in MUSK suggests that the level of MuSK phosphorylation is crucial for governing synaptic structure.

Hum Mutat 2020 03 25;41(3):619-631. Epub 2019 Nov 25.

Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK.

MUSK encodes the muscle-specific receptor tyrosine kinase (MuSK), a key component of the agrin-LRP4-MuSK-DOK7 signaling pathway, which is essential for the formation and maintenance of highly specialized synapses between motor neurons and muscle fibers. We report a patient with severe early-onset congenital myasthenic syndrome and two novel missense mutations in MUSK (p.C317R and p.A617V). Functional studies show that MUSK p.C317R, located at the frizzled-like cysteine-rich domain of MuSK, disrupts an integral part of MuSK architecture resulting in ablated MuSK phosphorylation and acetylcholine receptor (AChR) cluster formation. MUSK p.A617V, located at the kinase domain of MuSK, enhances MuSK phosphorylation resulting in anomalous AChR cluster formation. The identification and evidence for pathogenicity of MUSK mutations supported the initiation of treatment with β2-adrenergic agonists with a dramatic improvement of muscle strength in the patient. This work suggests uncharacterized mechanisms in which control of the precise level of MuSK phosphorylation is crucial in governing synaptic structure.
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http://dx.doi.org/10.1002/humu.23949DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7028094PMC
March 2020

Myasthenia Gravis: Pathogenic Effects of Autoantibodies on Neuromuscular Architecture.

Cells 2019 07 2;8(7). Epub 2019 Jul 2.

Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, 1090 Vienna, Austria.

Myasthenia gravis (MG) is an autoimmune disease of the neuromuscular junction (NMJ). Autoantibodies target key molecules at the NMJ, such as the nicotinic acetylcholine receptor (AChR), muscle-specific kinase (MuSK), and low-density lipoprotein receptor-related protein 4 (Lrp4), that lead by a range of different pathogenic mechanisms to altered tissue architecture and reduced densities or functionality of AChRs, reduced neuromuscular transmission, and therefore a severe fatigable skeletal muscle weakness. In this review, we give an overview of the history and clinical aspects of MG, with a focus on the structure and function of myasthenic autoantigens at the NMJ and how they are affected by the autoantibodies' pathogenic mechanisms. Furthermore, we give a short overview of the cells that are implicated in the production of the autoantibodies and briefly discuss diagnostic challenges and treatment strategies.
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http://dx.doi.org/10.3390/cells8070671DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6678492PMC
July 2019

Reduced muscle strength in ether lipid-deficient mice is accompanied by altered development and function of the neuromuscular junction.

J Neurochem 2017 12 25;143(5):569-583. Epub 2017 Sep 25.

Department of Pathobiology of the Nervous System, Center for Brain Research, Medical University of Vienna, Vienna, Austria.

Inherited deficiency in ether lipids, a subgroup of phospholipids whose biosynthesis needs peroxisomes, causes the fatal human disorder rhizomelic chondrodysplasia punctata. The exact roles of ether lipids in the mammalian organism and, therefore, the molecular mechanisms underlying the disease are still largely enigmatic. Here, we used glyceronephosphate O-acyltransferase knockout (Gnpat KO) mice to study the consequences of complete inactivation of ether lipid biosynthesis and documented substantial deficits in motor performance and muscle strength of these mice. We hypothesized that, probably in addition to previously described cerebellar abnormalities and myelination defects in the peripheral nervous system, an impairment of neuromuscular transmission contributes to the compromised motor abilities. Structurally, a morphologic examination of the neuromuscular junction (NMJ) in diaphragm muscle at different developmental stages revealed aberrant axonal branching and a strongly increased area of nerve innervation in Gnpat KO mice. Post-synaptically, acetylcholine receptor (AChR) clusters colocalized with nerve terminals within a widened endplate zone. In addition, we detected atypical AChR clustering, as indicated by decreased size and number of clusters following stimulation with agrin, in vitro. The turnover of AChRs was unaffected in ether lipid-deficient mice. Electrophysiological evaluation of the adult diaphragm indicated that although evoked potentials were unaltered in Gnpat KO mice, ether lipid deficiency leads to fewer spontaneous synaptic vesicle fusion events but, conversely, an increased post-synaptic response to spontaneous vesicle exocytosis. We conclude from our findings that ether lipids are essential for proper development and function of the NMJ and may, therefore, contribute to motor performance. Read the Editorial Highlight for this article on page 463.
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http://dx.doi.org/10.1111/jnc.14082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5725694PMC
December 2017

Motor neuronal repletion of the NMJ organizer, Agrin, modulates the severity of the spinal muscular atrophy disease phenotype in model mice.

Hum Mol Genet 2017 07;26(13):2377-2385

Department of Pathology and Cell Biology.

Spinal muscular atrophy (SMA) is a common and often fatal neuromuscular disorder caused by low levels of the Survival Motor Neuron (SMN) protein. Amongst the earliest detectable consequences of SMN deficiency are profound defects of the neuromuscular junctions (NMJs). In model mice these synapses appear disorganized, fail to mature and are characterized by poorly arborized nerve terminals. Given one role of the SMN protein in orchestrating the assembly of spliceosomal snRNP particles and subsequently regulating the alternative splicing of pre-mRNAs, a plausible link between SMN function and the distal neuromuscular SMA phenotype is an incorrectly spliced transcript or transcripts involved in establishing or maintaining NMJ structure. In this study, we explore the effects of one such transcript-Z+Agrin-known to be a critical organizer of the NMJ. We confirm that low SMN protein reduces motor neuronal levels of Z+Agrin. Repletion of this isoform of Agrin in the motor neurons of SMA model mice increases muscle fiber size, enhances the post-synaptic NMJ area, reduces the abnormal accumulation of intermediate filaments in nerve terminals of the neuromuscular synapse and improves the innervation of muscles. While these effects are independent of changes in SMN levels or increases in motor neuron numbers they nevertheless have a significant effect on the overall disease phenotype, enhancing mean survival in severely affected SMA model mice by ∼40%. We conclude that Agrin is an important target of the SMN protein and that mitigating NMJ defects may be one strategy in treating human spinal muscular atrophy.
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http://dx.doi.org/10.1093/hmg/ddx124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6074815PMC
July 2017

Fluorescence colocalization microscopy analysis can be improved by combining object-recognition with pixel-intensity-correlation.

Biotechnol J 2017 Jan 26;12(1). Epub 2016 Jul 26.

Dept. of Vascular Biology and Thrombosis Research, Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria.

The question whether two proteins interact with each other or whether a protein localizes to a certain region of the cell is often addressed with fluorescence microscopy and analysis of a potential colocalization of fluorescence markers. Since a mere visual estimation does not allow quantification of the degree of colocalization, different statistical methods of pixel-intensity correlation are commonly used to score it. We observed that these correlation coefficients are prone to false positive results and tend to show high values even for molecules that reside in different organelles. Our aim was to improve this type of analysis and we developed a novel method combining object-recognition based colocalization analysis with pixel-intensity correlation to calculate an object-corrected Pearson coefficient. We designed a macro for the Fiji-version of the software ImageJ and tested the performance systematically with various organelle markers revealing an improved robustness of our approach over classical methods. In order to prove that colocalization does not necessarily mean a physical interaction, we performed FRET (fluorescence resonance energy transfer) microscopy. This confirmed that non-interacting molecules can exhibit a nearly complete colocalization, but that they do not show any significant FRET signal in contrast to proteins that are bound to each other.
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http://dx.doi.org/10.1002/biot.201600332DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5244660PMC
January 2017

Neuromuscular synapse integrity requires linkage of acetylcholine receptors to postsynaptic intermediate filament networks via rapsyn-plectin 1f complexes.

Mol Biol Cell 2014 Dec 15;25(25):4130-49. Epub 2014 Oct 15.

Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories, University of Vienna, 1030 Vienna, Austria

Mutations in the cytolinker protein plectin lead to grossly distorted morphology of neuromuscular junctions (NMJs) in patients suffering from epidermolysis bullosa simplex (EBS)-muscular dystrophy (MS) with myasthenic syndrome (MyS). Here we investigated whether plectin contributes to the structural integrity of NMJs by linking them to the postsynaptic intermediate filament (IF) network. Live imaging of acetylcholine receptors (AChRs) in cultured myotubes differentiated ex vivo from immortalized plectin-deficient myoblasts revealed them to be highly mobile and unable to coalesce into stable clusters, in contrast to wild-type cells. We found plectin isoform 1f (P1f) to bridge AChRs and IFs via direct interaction with the AChR-scaffolding protein rapsyn in an isoform-specific manner; forced expression of P1f in plectin-deficient cells rescued both compromised AChR clustering and IF network anchoring. In conditional plectin knockout mice with gene disruption in muscle precursor/satellite cells (Pax7-Cre/cKO), uncoupling of AChRs from IFs was shown to lead to loss of postsynaptic membrane infoldings and disorganization of the NMJ microenvironment, including its invasion by microtubules. In their phenotypic behavior, mutant mice closely mimicked EBS-MD-MyS patients, including impaired body balance, severe muscle weakness, and reduced life span. Our study demonstrates that linkage to desmin IF networks via plectin is crucial for formation and maintenance of AChR clusters, postsynaptic NMJ organization, and body locomotion.
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http://dx.doi.org/10.1091/mbc.E14-06-1174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4263455PMC
December 2014

Global analysis of muscle-specific kinase signaling by quantitative phosphoproteomics.

Mol Cell Proteomics 2014 Aug 4;13(8):1993-2003. Epub 2014 Jun 4.

‖Center for Brain Research, Medical University of Vienna, Spitalgasse 4, 1090 Vienna, Austria; ‡‡Institute of Immunology, Medical University of Vienna, Lazarettgasse 19, 1090 Vienna, Austria.

The development of the neuromuscular synapse depends on signaling processes that involve protein phosphorylation as a crucial regulatory event. Muscle-specific kinase (MuSK) is the key signaling molecule at the neuromuscular synapse whose activity is required for the formation of a mature and functional synapse. However, the signaling cascade downstream of MuSK and the regulation of the different components are still poorly understood. In this study we used a quantitative phosphoproteomics approach to study the phosphorylation events and their temporal regulation downstream of MuSK. We identified a total of 10,183 phosphopeptides, of which 203 were significantly up- or down-regulated. Regulated phosphopeptides were classified into four different clusters according to their temporal profiles. Within these clusters we found an overrepresentation of specific protein classes associated with different cellular functions. In particular, we found an enrichment of regulated phosphoproteins involved in posttranscriptional mechanisms and in cytoskeletal organization. These findings provide novel insights into the complex signaling network downstream of MuSK and form the basis for future mechanistic studies.
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http://dx.doi.org/10.1074/mcp.M113.036087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4125732PMC
August 2014

Endosomal trafficking of the receptor tyrosine kinase MuSK proceeds via clathrin-dependent pathways, Arf6 and actin.

FEBS J 2013 Jul 23;280(14):3281-97. Epub 2013 May 23.

Center for Brain Research, Medical University of Vienna, Vienna, Austria.

Muscle-specific kinase (MuSK), a receptor tyrosine kinase, is the key player during the formation of the neuromuscular junction. Signal transduction events downstream of MuSK activation induce both pre- and postsynaptic differentiation, which, most prominently, includes the clustering of acetylcholine receptors at synaptic sites. More recently, regulated MuSK endocytosis and degradation have been implicated as crucial events for MuSK signalling activity, implicating a cross-talk between signalling and endocytosis. In the present study, we use a live imaging approach to study MuSK endocytosis. We find that MuSK is internalized via a clathrin-, dynamin-dependent pathway. MuSK is transported to Rab7-positive endosomes for degradation and recycled via Rab4- and Rab11-positive vesicles. MuSK activation by Dok7 mildly affects the localization of MuSK on the cell surface but has no effect on the rate of MuSK internalization. Interestingly, MuSK colocalizes with actin and Arf6 at the cell surface and during endosomal trafficking. Disruption of the actin cytoskeleton or the proper function of Arf6 concentrates MuSK in cell protrusions. Moreover, inhibition of Arf6 or cytoskeletal rearrangements impairs acetylcholine receptor clustering and phosphorylation. These results suggest that MuSK uses both classical and nonclassical endosomal pathways that involve a variety of different components of the endosomal machinery.
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http://dx.doi.org/10.1111/febs.12309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3806275PMC
July 2013

CLP1 links tRNA metabolism to progressive motor-neuron loss.

Nature 2013 Mar 10;495(7442):474-80. Epub 2013 Mar 10.

IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna 1030, Austria.

CLP1 was the first mammalian RNA kinase to be identified. However, determining its in vivo function has been elusive. Here we generated kinase-dead Clp1 (Clp1(K/K)) mice that show a progressive loss of spinal motor neurons associated with axonal degeneration in the peripheral nerves and denervation of neuromuscular junctions, resulting in impaired motor function, muscle weakness, paralysis and fatal respiratory failure. Transgenic rescue experiments show that CLP1 functions in motor neurons. Mechanistically, loss of CLP1 activity results in accumulation of a novel set of small RNA fragments, derived from aberrant processing of tyrosine pre-transfer RNA. These tRNA fragments sensitize cells to oxidative-stress-induced p53 (also known as TRP53) activation and p53-dependent cell death. Genetic inactivation of p53 rescues Clp1(K/K) mice from the motor neuron loss, muscle denervation and respiratory failure. Our experiments uncover a mechanistic link between tRNA processing, formation of a new RNA species and progressive loss of lower motor neurons regulated by p53.
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http://dx.doi.org/10.1038/nature11923DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3674495PMC
March 2013

A mutation causes MuSK reduced sensitivity to agrin and congenital myasthenia.

PLoS One 2013 9;8(1):e53826. Epub 2013 Jan 9.

Inserm, UMRS 975, UPMC, Institut du Cerveau et de la Moelle épinière, Groupe Hospitalier Pitié-Salpêtrière, Paris, France.

Congenital myasthenic syndromes (CMSs) are a heterogeneous group of genetic disorders affecting neuromuscular transmission. The agrin/muscle-specific kinase (MuSK) pathway is critical for proper development and maintenance of the neuromuscular junction (NMJ). We report here an Iranian patient in whom CMS was diagnosed since he presented with congenital and fluctuating bilateral symmetric ptosis, upward gaze palsy and slowly progressive muscle weakness leading to loss of ambulation. Genetic analysis of the patient revealed a homozygous missense mutation c.2503A>G in the coding sequence of MUSK leading to the p.Met835Val substitution. The mutation was inherited from the two parents who were heterozygous according to the notion of consanguinity. Immunocytochemical and electron microscopy studies of biopsied deltoid muscle showed dramatic changes in pre- and post-synaptic elements of the NMJs. These changes induced a process of denervation/reinnervation in native NMJs and the formation, by an adaptive mechanism, of newly formed and ectopic NMJs. Aberrant axonal outgrowth, decreased nerve terminal ramification and nodal axonal sprouting were also noted. In vivo electroporation of the mutated MuSK in a mouse model showed disorganized NMJs and aberrant axonal growth reproducing a phenotype similar to that observed in the patient's biopsy specimen. In vitro experiments showed that the mutation alters agrin-dependent acetylcholine receptor aggregation, causes a constitutive activation of MuSK and a decrease in its agrin- and Dok-7-dependent phosphorylation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0053826PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3541344PMC
July 2013

The formation of complex acetylcholine receptor clusters requires MuSK kinase activity and structural information from the MuSK extracellular domain.

Mol Cell Neurosci 2012 Apr 23;49(4):475-86. Epub 2011 Dec 23.

Center for Brain Research, Medical University of Vienna, Spitalgasse 4, 1090 Vienna, Austria.

Efficient synaptic transmission at the neuromuscular junction (NMJ) requires the topological maturation of the postsynaptic apparatus from an oval acetylcholine receptor (AChR)-rich plaque into a complex pretzel-shaped array of branches. However, compared to NMJ formation very little is known about the mechanisms that regulate NMJ maturation. Recently the process of in vivo transformation from plaque into pretzel has been reproduced in vitro by culturing myotubes aneurally on laminin-coated substrate. It was proposed that the formation of complex AChR clusters is regulated by a MuSK-dependent muscle intrinsic program. To elucidate the structure-function role of MuSK in the aneural maturation of AChR pretzels, we used muscle cell lines expressing MuSK mutant and chimeric proteins. Here we report, that besides its role during agrin-induced AChR clustering, MuSK kinase activity is also necessary for substrate-dependent cluster formation. Constitutive-active MuSK induces larger AChR clusters, a faster cluster maturation on laminin and increases the anchorage of AChRs to the cytoskeleton compared to MuSK wild-type. In addition, we find that the juxtamembrane region of MuSK, which has previously been shown to regulate agrin-induced AChR clustering, is unable to induce complex AChR clusters on laminin substrate. Most interestingly, MuSK kinase activity is not sufficient for laminin-dependent AChR cluster formation since the MuSK ectodomain is also required suggesting a so far undiscovered instructive role for the extracellular domain of MuSK.
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http://dx.doi.org/10.1016/j.mcn.2011.12.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3359500PMC
April 2012

Rin-like, a novel regulator of endocytosis, acts as guanine nucleotide exchange factor for Rab5a and Rab22.

Biochim Biophys Acta 2011 Jun 17;1813(6):1198-210. Epub 2011 Mar 17.

Center for Brain Research, Medical University of Vienna, Spitalgasse 4, A-1090 Vienna, Austria.

RIN proteins serve as guanine nucleotide exchange factors for Rab5a. They are characterized by the presence of a RIN homology domain and a C-terminal Vps9 domain. Currently three family members have been described and analyzed. Here we report the identification of a novel RIN family member, Rin-like (Rinl), that represents a new interaction partner of the receptor tyrosine kinase MuSK, which is an essential key regulator of neuromuscular synapse development. Rinl is localized to neuromuscular synapses but shows the highest expression in thymus and spleen. Rinl preferentially binds to nucleotide-free Rab5a and catalyzes the exchange of GDP for GTP. Moreover, Rinl also binds GDP-bound Rab22 and increases the GDP/GTP exchange implicating Rinl in endocytotic processes regulated by Rab5a and Rab22. Interestingly, Rinl shows a higher catalytic rate for Rab22 compared to Rab5a. Rinl is closely associated with the cytoskeleton and thus contributes to the spatial control of Rab5a and Rab22 signaling at actin-positive compartments. Most importantly, overexpression of Rinl affects fluid-phase as well as EGFR endocytosis.
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http://dx.doi.org/10.1016/j.bbamcr.2011.03.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3096779PMC
June 2011

Aberrant development of neuromuscular junctions in glycosylation-defective Large(myd) mice.

Neuromuscul Disord 2009 May 5;19(5):366-78. Epub 2009 Apr 5.

Center for Brain Research, Medical University of Vienna, Vienna, Austria.

Mice deficient in the glycosyltransferase Large are characterized by severe muscle and central nervous system abnormalities. In this study, we show that the formation and maintenance of neuromuscular junctions in Large(myd) mice are greatly compromised. Neuromuscular junctions are not confined to the muscle endplate zone but are widely spread and are frequently accompanied by exuberant nerve sprouting. Nerve terminals are highly fragmented and binding of alpha-bungarotoxin to postsynaptic acetylcholine receptors (AChRs) is greatly reduced. In vitro, Large(myd) myotubes are responsive to agrin but produce aberrant AChR clusters, which are larger in area and less densely packed with AChRs. In addition, AChR expression on the cell surface is diminished suggesting that AChR assembly or transport is defective. These results together with the finding that O-linked glycosylation at neuromuscular junctions of Large(myd) mice is compromised indicate that the action of Large is necessary for proper neuromuscular junction development.
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http://dx.doi.org/10.1016/j.nmd.2009.02.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2958302PMC
May 2009

Rescuing Z+ agrin splicing in Nova null mice restores synapse formation and unmasks a physiologic defect in motor neuron firing.

Proc Natl Acad Sci U S A 2009 Mar 12;106(9):3513-8. Epub 2009 Feb 12.

Laboratory of Molecular Neuro-Oncology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.

Synapse formation at the neuromuscular junction (NMJ) requires an alternatively spliced variant of agrin (Z(+) agrin) that is produced only by neurons. Here, we show that Nova1 and Nova2, neuron-specific splicing factors identified as targets in autoimmune motor disease, are essential regulators of Z(+) agrin. Nova1/Nova2 double knockout mice are paralyzed and fail to cluster AChRs at the NMJ, and breeding them with transgenic mice constitutively expressing Z(+) agrin in motor neurons rescued AChR clustering. Surprisingly, however, these rescued mice remained paralyzed, while electrophysiologic studies demonstrated that the motor axon and synapse were functional-spontaneous and evoked recordings revealed synaptic transmission and muscle contraction. These results point to a proximal defect in motor neuron firing in the absence of Nova and reveal a previously unsuspected role for RNA regulation in the physiologic activation of motor neurons.
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http://dx.doi.org/10.1073/pnas.0813112106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2642475PMC
March 2009

Essential role of B-Raf in oligodendrocyte maturation and myelination during postnatal central nervous system development.

J Cell Biol 2008 Mar;180(5):947-55

Max F. Perutz Laboratories, University of Vienna, 1030 Vienna, Austria.

Mutations in the extracellular signal-regulated kinase (ERK) pathway, particularly in the mitogen-activated protein kinase/ERK kinase (MEK) activator B-Raf, are associated with human tumorigenesis and genetic disorders. Hence, B-Raf is a prime target for molecule-based therapies, and understanding its essential biological functions is crucial for their success. B-Raf is expressed preferentially in cells of neuronal origin. Here, we show that in mice, conditional ablation of B-Raf in neuronal precursors leads to severe dysmyelination, defective oligodendrocyte differentiation, and reduced ERK activation in brain. Both B-Raf ablation and chemical inhibition of MEK impair oligodendrocyte differentiation in vitro. In glial cell cultures, we find B-Raf in a complex with MEK, Raf-1, and kinase suppressor of Ras. In B-Raf-deficient cells, more Raf-1 is recruited to MEK, yet MEK/ERK phosphorylation is impaired. These data define B-Raf as the rate-limiting MEK/ERK activator in oligodendrocyte differentiation and myelination and have implications for the design and use of Raf inhibitors.
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http://dx.doi.org/10.1083/jcb.200709069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2265404PMC
March 2008

Phosphoinositide 3-kinase acts through RAC and Cdc42 during agrin-induced acetylcholine receptor clustering.

Dev Neurobiol 2007 Jul;67(8):1047-58

Center for Brain Research, Medical University of Vienna, Vienna, Austria.

The formation of the neuromuscular junction (NMJ) is regulated by the nerve-derived heparan sulfate proteoglycan agrin and the muscle-specific kinase MuSK. Agrin induces a signal transduction pathway via MuSK, which promotes the reorganization of the postsynaptic muscle membrane. Activation of MuSK leads to the phosphorylation and redistribution of acetylcholine receptors (AChRs) and other postsynaptic proteins to synaptic sites. The accumulation of high densities of AChRs at postsynaptic regions represents a hallmark of NMJ formation and is required for proper NMJ function. Here we show that phosphoinositide 3-kinase (PI3-K) represents a component of the agrin/MuSK signaling pathway. Muscle cells treated with specific PI3-K inhibitors are unable to form full-size AChR clusters in response to agrin and AChR phosphorylation is reduced. Moreover, agrin-induced activation of Rac and Cdc42 is impaired in the presence of PI3-K inhibitors. PI3-K is localized to the postsynaptic muscle membrane consistent with a role during agrin/MuSK signaling. These results put PI3-K downstream of MuSK as regulator of AChR phosphorylation and clustering. Its role during agrin-stimulated Rac and Cdc42 activation suggests a critical function during cytoskeletal reorganizations, which lead to the redistribution of actin-anchored AChRs.
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http://dx.doi.org/10.1002/dneu.20371DOI Listing
July 2007

[Pathophysiological characterization of congenital myasthenic syndromes: the example of mutations in the MUSK gene].

J Soc Biol 2005 ;199(1):61-77

INSERM U582 & IFR 14, Institut de Myologie, Hôpital de la Salpêtrière et Université Pierre et Marie Curie, Paris, France.

Congenital myasthenic syndromes (CMS) are rare genetic diseases affecting the neuromuscular junction (NMJ) and are characterized by a dysfunction of the neurotransmission. They are heterogeneous at their pathophysiological level and can be classified in three categories according to their presynaptic, synaptic and postsynaptic origins. We report here the first case of a human neuromuscular transmission dysfunction due to mutations in the gene encoding a postsynaptic molecule, the muscle-specific receptor tyrosine kinase (MuSK). Gene analysis identified two heteroallelic mutations, a frameshift mutation (c.220insC) and a missense mutation (V790M). The muscle biopsy showed dramatic pre- and postsynaptic structural abnormalities of the neuromuscular junction and severe decrease in acetylcholine receptor (AChR) epsilon-subunit and MuSK expression. In vitro and in vivo expression experiments were performed using mutant MuSK reproducing the human mutations. The frameshift mutation led to the absence of MuSK expression. The missense mutation did not affect MuSK catalytic kinase activity but diminished expression and stability of MuSK leading to decreased agrin-dependent AChR aggregation, a critical step in the formation of the neuromuscular junction. In electroporated mouse muscle, overexpression of the missense mutation induced, within a week, a phenotype similar to the patient muscle biopsy: a severe decrease in synaptic AChR and an aberrant axonal outgrowth. These results strongly suggest that the missense mutation, in the presence of a null mutation on the other allele, is responsible for the dramatic synaptic changes observed in the patient.
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http://dx.doi.org/10.1051/jbio:2005008DOI Listing
October 2005

MUSK, a new target for mutations causing congenital myasthenic syndrome.

Hum Mol Genet 2004 Dec 20;13(24):3229-40. Epub 2004 Oct 20.

INSERM U582 & IFR Cur, Muscle, Vaisseaux, Institut de Myologie, Hôpital de la Salpêtrière and Université Pierre et Marie Curie, Paris, France.

We report the first case of a human neuromuscular transmission dysfunction due to mutations in the gene encoding the muscle-specific receptor tyrosine kinase (MuSK). Gene analysis identified two heteroallelic mutations, a frameshift mutation (c.220insC) and a missense mutation (V790M). The muscle biopsy showed dramatic pre- and postsynaptic structural abnormalities of the neuromuscular junction and severe decrease in acetylcholine receptor (AChR) epsilon-subunit and MuSK expression. In vitro and in vivo expression experiments were performed using mutant MuSK reproducing the human mutations. The frameshift mutation led to the absence of MuSK expression. The missense mutation did not affect MuSK catalytic kinase activity but diminished expression and stability of MuSK leading to decreased agrin-dependent AChR aggregation, a critical step in the formation of the neuromuscular junction. In electroporated mouse muscle, overexpression of the missense mutation induced, within a week, a phenotype similar to the patient muscle biopsy: a severe decrease in synaptic AChR and an aberrant axonal outgrowth. These results strongly suggest that the missense mutation, in the presence of a null mutation on the other allele, is responsible for the dramatic synaptic changes observed in the patient.
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http://dx.doi.org/10.1093/hmg/ddh333DOI Listing
December 2004

Restoration of synapse formation in Musk mutant mice expressing a Musk/Trk chimeric receptor.

Development 2002 Dec;129(23):5449-60

Molecular Neurobiology Program, Skirball Institute, NYU Medical School, NY 10016, USA.

Mice lacking Musk, a muscle-specific receptor tyrosine kinase that is activated by agrin, fail to form neuromuscular synapses and consequently die at birth because of their failure to move or breathe. We produced mice that express a chimeric receptor, containing the juxtamembrane region of Musk and the kinase domain of TrkA, selectively in muscle, and we crossed this transgene into Musk mutant mice. Expression of this chimeric receptor restores presynaptic and postsynaptic differentiation, including the formation of nerve terminal arbors, synapse-specific transcription, and clustering of postsynaptic proteins, allowing Musk mutant mice to move, breathe and survive as adults. These results show that the juxtamembrane region of Musk, including a single phosphotyrosine docking site, even in the context of a different kinase domain, is sufficient to activate the multiple pathways leading to presynaptic and postsynaptic differentiation in vivo. In addition, we find that Musk protein can be clustered at synaptic sites, even if Musk mRNA is expressed uniformly in muscle. Moreover, acetylcholine receptor clustering and motor terminal branching are restored in parallel, indicating that the extent of presynaptic differentiation is matched to the extent of postsynaptic differentiation.
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http://dx.doi.org/10.1242/dev.00112DOI Listing
December 2002
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