Auto-antibodies to the receptor tyrosine kinase MuSK in patients with myasthenia gravis without acetylcholine receptor antibodies.

Myasthenia gravis (MG) is an antibody-mediated autoimmune disease of the neuromuscular junction. In approximately 80% of patients, auto-antibodies to the muscle nicotinic acetylcholine receptor (AChR) are present. These antibodies cause loss of AChR numbers and function, and lead to failure of neuromuscular transmission with muscle weakness. The pathogenic mechanisms acting in the 20% of patients with generalized MG who are seronegative for AChR-antibodies (AChR-Ab) have not been elucidated, but there is evidence that they also have an antibody-mediated disorder, with the antibodies directed towards another, previously unidentified muscle-surface-membrane target. Here we show that 70% of AChR-Ab-seronegative MG patients, but not AChR-Ab-seropositive MG patients, have serum auto-antibodies against the muscle-specific receptor tyrosine kinase, MuSK. MuSK mediates the agrin-induced clustering of AChRs during synapse formation, and is also expressed at the mature neuromuscular junction. The MuSK antibodies were specific for the extracellular domains of MuSK expressed in transfected COS7 cells and strongly inhibited MuSK function in cultured myotubes. Our results indicate the involvement of MuSK antibodies in the pathogenesis of AChR-Ab-seronegative MG, thus defining two immunologically distinct forms of the disease. Measurement of MuSK antibodies will substantially aid diagnosis and clinical management.

MAGI-1c: a synaptic MAGUK interacting with muSK at the vertebrate neuromuscular junction.

The muscle-specific receptor tyrosine kinase (MuSK) forms part of a receptor complex, activated by nerve-derived agrin, that orchestrates the differentiation of the neuromuscular junction (NMJ). The molecular events linking MuSK activation with postsynaptic differentiation are not fully understood. In an attempt to identify partners and/or effectors of MuSK, cross-linking and immunopurification experiments were performed in purified postsynaptic membranes from the Torpedo electrocyte, a model system for the NMJ. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis was conducted on both cross-link products, and on the major peptide coimmunopurified with MuSK; this analysis identified a polypeptide corresponding to the COOH-terminal fragment of membrane-associated guanylate kinase (MAGUK) with inverted domain organization (MAGI)-1c. A bona fide MAGI-1c (150 kD) was detected by Western blotting in the postsynaptic membrane of Torpedo electrocytes, and in a high molecular mass cross-link product of MuSK. Immunofluorescence experiments showed that MAGI-1c is localized specifically at the adult rat NMJ, but is absent from agrin-induced acetylcholine receptor clusters in myotubes in vitro. In the central nervous system, MAGUKs play a primary role as scaffolding proteins that organize cytoskeletal signaling complexes at excitatory synapses. Our data suggest that a protein from the MAGUK family is involved in the MuSK signaling pathway at the vertebrate NMJ.

Arachidonic acid causes sudden death in rabbits.

Injection of sodium arachidonate (1.4 milligrams per kilogram) into the marginal ear veins of rabbits caused death within 3 minutes. Histological examination showed platelet thrombi in the microvasculature of the lungs. Rabbits were protected from the lethal effects of arachidonic acid by pretreatment with aspirin. Fatty acids closely related to arachidonic acid did not cause death.

Primary cultures of mouse spinal cord express the neonatal isoform of the inhibitory glycine receptor.

Expression of the inhibitory glycine receptor complex was investigated in primary cultures of fetal mouse spinal cord using sensitive immunomethods. In these cells, glycine receptor is predominantly of the neonatal isoform characterized by a low affinity for the antagonist strychnine. It contains a ligand binding subunit that differs from that of the adult receptor in antigenic epitopes and apparent molecular weight. Whereas in vivo the neonatal receptor isoform is completely replaced by the adult isoform within 3 weeks after birth, this exchange of subtypes is not seen in culture. The increased expression of the cytoplasmic glycine receptor-associated polypeptide of 93 kd occurring after birth is also seen under culture conditions. Purification of glycine receptor from cultures yielded polypeptides of 49 kd and 93 kd, suggesting that the membrane-spanning core of the neonatal receptor may be a homooligomer composed of 49 kd subunits. About half of the 49 kd subunit is cleaved by trypsinization of the cultures, indicating a predominant cell surface localization of the receptor. Pulse-labeling experiments revealed the 49 kd subunit to be a metabolically stable glycoprotein (half-life approximately 2 days). After its synthesis, a transition time of 30-45 min is required for acquisition of a strychnine binding conformation.

Developmental regulation of highly active alternatively spliced forms of agrin.

Agrin is an extracellular matrix protein involved in clustering acetylcholine receptors during development of the neuromuscular junction. We have previously shown that alternative splicing at three sites generates multiple forms of rat agrin and that a novel 8 amino acid insert is the most important in determining biological activity. In the present study we have examined the expression of agrin during development with particular emphasis on determining the tissue distribution of the splicing variants at each site. Our principal observation is that the variants containing the sequence most responsible for biological activity are expressed exclusively in neural tissue and that their expression is highly regulated during development. We also show that muscle expresses less active forms and that agrin immunoreactivity during synaptogenesis is initially not limited to synaptic sites, but becomes progressively restricted to the synapse as development proceeds.

The ability of agrin to cluster AChRs depends on alternative splicing and on cell surface proteoglycans.

Agrin, which induces acetylcholine receptor (AChR) clustering at the developing neuromuscular synapse, occurs in multiple forms generated by alternative splicing. Some of these isoforms are specific to the nervous system; others are expressed in both neural and nonneural tissues, including muscle. We have compared the AChR clustering activity of agrin forms varying at each of the three identified splicing sites, denoted x, y, and z. Agrin isoforms were assayed by applying either transfected COS cells, with agrin bound to their surfaces, or soluble agrin to myotubes of the C2 muscle line, or of two variant lines having defective proteoglycans. Dramatic differences in activity were seen between z site isoforms and lesser differences between y site isoforms. The most active agrin forms contained splicing inserts of 4 amino acids at the y site and 8 amino acids at the z site. These forms are found exclusively in neural tissue. All forms were active on C2 myotubes in cell-attached assays, but muscle forms were less active than neural forms. AChR clustering activity of all agrin forms was decreased when assayed on the proteoglycan-deficient lines, suggesting that proteoglycans may help mediate the action of agrin. As neural agrin forms are more active than muscle forms, they are likely to play a primary role in synaptogenesis.

RNA splicing regulates agrin-mediated acetylcholine receptor clustering activity on cultured myotubes.

Agrin is a component of the synaptic basal lamina that induces the clustering of acetylcholine receptors (AChRs) on muscle fibers. A region near the carboxyl terminus of the protein exists in four forms that are generated by alternative RNA splicing. All four alternatively spliced forms of agrin are active in inducing AChR clusters on rat primary and C2-derived muscle fibers. In contrast, only two forms of the protein, each containing an 8 amino acid insert, are capable of inducing clusters on myotubes of S27 cells, a C2 variant that has defective proteoglycans. These two forms are also most active in inducing clusters on chick myotubes. This pattern of differential activity suggests that RNA splicing of agrin transcripts and interactions with proteoglycans or other components of basal lamina have important roles in regulating the localization of neurotransmitter receptors at synaptic sites.

Agrin and the organization of the neuromuscular junction.

Agrin is a component of the synaptic extracellular matrix and may regulate the organization of acetylcholine receptors and other synaptic molecules in both synapse regeneration and development. Analyses of cDNAs encoding agrin define a number of structural domains, including regions of homology to laminin, Kazal protease inhibitors, and epidermal growth factor repeats.

Delayed synapsing muscles are more severely affected in an experimental model of MuSK-induced myasthenia gravis.

Myasthenia gravis can be induced in mice by injecting the extracellular domain of rat muscle-specific kinase (MuSK), a transmembrane receptor tyrosine kinase involved in agrin signaling at the neuromuscular junction. About 5-10% of human myasthenia gravis patients have autoantibodies against MuSK. Here we have examined mouse neuromuscular junctions following MuSK immunization in two groups of muscles that can be distinguished on the basis of the timing of neuromuscular synaptogenesis and their response to perturbation of agrin signaling. We used confocal microscopy to characterize the distribution and expression of nicotinic acetylcoline receptors and of two presynaptic makers, neurofilament protein and synaptophysin. We observed disruption of neuromuscular junctions in all muscles examined in this model of myasthenia gravis. However delayed-synapsing muscles, including the diaphragm, sternomastoid and tibialis posterior, were significantly more severely affected than fast-synapsing muscles, including the intercostal, adductor longus and tibialis anterior. These results suggest a basis for the differential susceptibility of muscles in different classes of myasthenia gravis patients, including patients with autoantibodies against MuSK.

The 93 kDa protein gephyrin and tubulin associated with the inhibitory glycine receptor are phosphorylated by an endogenous protein kinase.

The 93 kDa protein gephyrin is a tubulin binding peripheral membrane protein that is associated with the inhibitory glycine receptor and has been implicated in its anchoring at central synapses. Here, we demonstrate that gephyrin as well as co-purifying tubulin are phosphorylated by a kinase activity which is endogenous to highly purified glycine receptor preparations. This kinase phosphorylates serine and threonine residues and utilizes ATP, but not GTP, as phosphate donor. Its activity is not affected by various activators and/or inhibitors of cyclic nucleotide-dependent kinases, calcium/calmodulin-dependent kinases, or protein kinase C. A five-fold stimulation of kinase activity was, however, observed in the presence of poly-lysine. Phosphorylation of gephyrin and/or tubulin might regulate receptor/cytoskeleton interactions at postsynaptic membrane specializations.

Modulation by NMDA Receptor Antagonists of Glycine Receptor Isoform Expression in Cultured Spinal Cord Neurons.

Two developmentally regulated isoforms of the inhibitory glycine receptor harbouring different alpha subunit variants, GlyRN (neonatal) and GlyRA (adult), have previously been identified in rodent spinal cord. Primary cultures of embyronic spinal neurons, however, express predominantly GlyRN. Here, N-methyl-d-aspartate (NMDA) receptor antagonists were found to significantly increase glycine receptor levels in mouse spinal cord cultures. In the presence of 2-amino-5-phosphonovalerate or MK-801 (dizocilpine), both GlyRN and GlyRA contents were elevated, as revealed by isoform-selective immunoassays and amplification of corresponding alpha subunit transcripts by the polymerase chain reaction. This effect of NMDA receptor antagonists was restricted to a 'sensitive' period within the second week after plating. Apparently, NMDA receptor-mediated glutamate neurotoxicity prevented GlyRA accumulation under standard culture conditions. Our data indicate that neuronal maturation in cell culture depends on conditions which minimize cell death resulting from glutamate release into the culture medium.

Calcium-dependent maintenance of agrin-induced postsynaptic specializations.

Although much progress has been made in understanding synapse formation, little is known about the mechanisms underlying synaptic maintenance and loss. The formation of agrin-induced AChR clusters on cultured myotubes requires both activation of the receptor tyrosine kinase MuSK and intracellular calcium fluxes. Here, we provide evidence that such AChR clusters are maintained by agrin/MuSK-induced intracellular calcium fluxes. Clamping intracellular calcium fluxes after AChR clusters have formed leads to rapid MuSK and AChR tyrosine dephosphorylation and cluster dispersal, even in the continued presence of agrin. Both the dephosphorylation and the dispersal are inhibited by the tyrosine phosphatase inhibitor pervanadate. In contrast, clamping intracellular calcium at the time of initial agrin stimulation has no effect on agrin-induced MuSK or AChR phosphorylation, but blocks AChR cluster formation. These findings suggest an avenue by which postsynaptic stability can be regulated by modification of intracellular signaling pathways that are distinct from those used during synapse formation.

Physiological significance of anthocyanins during autumnal leaf senescence.

The light screen hypothesis states that foliar anthocyanins shade the photosynthetic apparatus from excess light. In this paper we extend the light screen hypothesis, postulating that plant species at risk of photoinhibitory conditions during autumnal leaf senescence often utilize anthocyanins to protect the photosynthetic apparatus during the period of nutrient resorption. When senescence-related photosynthetic instabilities are compounded by other environmental stresses, particularly low temperature, severe photoinhibition may result in reduced resorption of critical foliar nutrients, which can significantly affect plant fitness. There is evidence that environments where low and often freezing temperatures are common in autumn selectively favor the production of anthocyanins in senescing foliage. The stimuli for, and the timing and location of, autumnal anthocyanin production are all consistent with a photoprotective role for these pigments in senescing leaves. Furthermore, differences in nitrogen allocation strategies between early and late successional species appear to affect photosynthetic stability during leaf senescence, resulting in a reduced need for foliar autumnal anthocyanins in many early successional plants. The ecological and physiological evidence presented in this paper suggest that, for many deciduous species, the production of anthocyanins provides effective photoprotection during the critical period of foliar nutrient resorption.

Resistance to the birch leafminer Fenusa pusilla (Hymenoptera: Tenthredinidae) within the genus Betula.

Thirteen Betula species were tested for resistance to the birch leafminer, Femusa pusilla (Lepeletier), using no-choice assays. Birch leafminers were able to oviposit into expanding leaves of all Betula individuals tested. Larvae did not survive within any of the tested individuals of three species, B. alleghaniensis (Britt.), B. grossa (S. & Z.), and B. lenta (L.). Leafminer eggs deposited into the leaves of these species hatched, and larvae fed for a short period before dying. These three species were classified as highly resistant to birch leafminer, based on very low percent of mines (0.6-3.1%) with a diameter >3 mm. Eight species, B. papyrifera (Marsh), B. pendula (Roth), B. turkestanica (Litvin), B. glandulifira (Regal), B. ermanii (Cham.), B. platyphylla variety japonica [(Miq.) Hara], B. populifolia (Marsh) and B. maximowicziana (Regal) were classified as susceptible, with percent of mines >3 mm diameter of 87-94%. Two species, B. costata (Trautv.) and B. davurica (Pall.), displayed intermediate and variable resistance. B. davurica exhibited a mechanism of resistance not observed in the other species, Eggs oviposited into the leaves of resistant B. davurica individuals became surrounded by an area of discolored and necrotic tissue, and died. This response resembles the programmed cell death associated with a hypersensitive response.