The mechanosensitive ion channel ASIC2 mediates both proprioceptive sensing and spinal alignment.

By translating mechanical forces into molecular signals, proprioceptive neurons provide the CNS with information on muscle length and tension, which is necessary to control posture and movement. However, the identities of the molecular players that mediate proprioceptive sensing are largely unknown. Here, we confirm the expression of the mechanosensitive ion channel ASIC2 in proprioceptive sensory neurons. By combining in vivo proprioception-related functional tests with ex vivo electrophysiological analyses of muscle spindles, we showed that mice lacking Asic2 display impairments in muscle spindle responses to stretch and motor coordination tasks. Finally, analysis of skeletons of Asic2 loss-of-function mice revealed a specific effect on spinal alignment. Overall, we identify ASIC2 as a key component in proprioceptive sensing and a regulator of spine alignment.

Degeneration of muscle spindles in a murine model of Pompe disease.

Pompe disease is a debilitating medical condition caused by a functional deficiency of lysosomal acid alpha-glucosidase (GAA). In addition to muscle weakness, people living with Pompe disease experience motor coordination deficits including an instable gait and posture. We reasoned that an impaired muscle spindle function might contribute to these deficiencies and therefore analyzed proprioception as well as muscle spindle structure and function in 4- and 8-month-old Gaa-/- mice. Gait analyses showed a reduced inter-limb and inter-paw coordination in Gaa-/- mice. Electrophysiological analyses of single-unit muscle spindle proprioceptive afferents revealed an impaired sensitivity of the dynamic and static component of the stretch response. Finally, a progressive degeneration of the sensory neuron and of the intrafusal fibers was detectable in Gaa-/- mice. We observed an increased abundance and size of lysosomes, a fragmentation of the inner and outer connective tissue capsule and a buildup of autophagic vacuoles in muscle spindles from 8-month-old Gaa-/- mice, indicating lysosomal defects and an impaired autophagocytosis. These results demonstrate a structural and functional degeneration of muscle spindles and an altered motor coordination in Gaa-/- mice. Similar changes could contribute to the impaired motor coordination in patients living with Pompe disease.

Severe congenital myasthenic syndromes caused by agrin mutations affecting secretion by motoneurons.

Congenital myasthenic syndromes (CMS) are predominantly characterized by muscle weakness and fatigability and can be caused by a variety of mutations in genes required for neuromuscular junction formation and maintenance. Among them, AGRN encodes agrin, an essential synaptic protein secreted by motoneurons. We have identified severe CMS patients with uncharacterized p.R1671Q, p.R1698P and p.L1664P mutations in the LG2 domain of agrin. Overexpression in primary motoneurons cultures in vitro and in chick spinal motoneurons in vivo revealed that the mutations modified agrin trafficking, leading to its accumulation in the soma and/or in the axon. Expression of mutant agrins in cultured cells demonstrated accumulation of agrin in the endoplasmic reticulum associated with induction of unfolded protein response (UPR) and impaired secretion in the culture medium. Interestingly, evaluation of the specific activity of individual agrins on AChR cluster formation indicated that when secreted, mutant agrins retained a normal capacity to trigger the formation of AChR clusters. To confirm agrin accumulation and secretion defect, iPS cells were derived from a patient and differentiated into motoneurons. Patient iPS-derived motoneurons accumulated mutant agrin in the soma and increased XBP1 mRNA splicing, suggesting UPR activation. Moreover, co-cultures of patient iPS-derived motoneurons with myotubes confirmed the deficit in agrin secretion and revealed a reduction in motoneuron survival. Altogether, we report the first mutations in AGRN gene that specifically affect agrin secretion by motoneurons. Interestingly, the three patients carrying these mutations were initially suspected of spinal muscular atrophy (SMA). Therefore, in the presence of patients with a clinical presentation of SMA but without mutation in the SMN1 gene, it can be worth to look for mutations in AGRN.

The effect of methocarbamol and mexiletine on murine muscle spindle function.

INTRODUCTION/AIMS: The muscle relaxant methocarbamol and the antimyotonic drug mexiletine are widely used for the treatment of muscle spasms, myotonia, and pain syndromes. To determine whether these drugs affect muscle spindle function, we studied their effect on the resting discharge and on stretch-induced action potential frequencies of proprioceptive afferent neurons. METHODS: Single unit action potential frequencies of proprioceptive afferents from muscle spindles in the murine extensor digitorum longus muscle of adult C57BL/6J mice were recorded under resting conditions and during ramp-and-hold stretches. Maximal tetanic force of the same muscle after direct stimulation was determined. High-resolution confocal microscopy analysis was performed to determine the distribution of Nav 1.4 channels, a potential target for both drugs. RESULTS: Methocarbamol and mexiletine inhibited the muscle spindle resting discharge in a dose-dependent manner with IC50 values around 300 µM and 6 µM, respectively. With increasing concentrations of both drugs, the response to stretch was also affected, with the static sensitivity first followed by the dynamic sensitivity. At high concentrations, both drugs completely blocked muscle spindle afferent output. Both drugs also reversibly reduced the specific force of the extensor digitorum longus muscle after tetanic stimulation. Finally, we present evidence for the presence and specific localization of the voltage-gated sodium channel Nav 1.4 in intrafusal fibers. DISCUSSION: In this study we demonstrate that both muscle relaxants affect muscle spindle function, suggesting impaired proprioception as a potential side effect of both drugs. Moreover, our results provide additional evidence of a peripheral activity of methocarbamol and mexiletine.

Autoantibodies to cortactin and agrin in sera of patients with myasthenia gravis.

Autoantibodies against agrin and cortactin have been described in patients with myasthenia gravis. To further validate and characterize these autoantibodies, sera and/or plasma exchange material of 135 patients with myasthenia gravis were screened for anti-agrin or anti-cortactin autoantibodies. Autoantibodies against cortactin were detected in three patients and two controls and could be confirmed by cell-based assays using cortactin-transfected human embryonic kidney cells in both controls and one patient, but were not detectable in follow-up sera of the three patients. We did not detect any autoantibodies against agrin. The clinical phenotype of anti-cortactin-positive patients varied, arguing against a relevant pathogenic role.

Muscle spindle function in healthy and diseased muscle.

Almost every muscle contains muscle spindles. These delicate sensory receptors inform the central nervous system (CNS) about changes in the length of individual muscles and the speed of stretching. With this information, the CNS computes the position and movement of our extremities in space, which is a requirement for motor control, for maintaining posture and for a stable gait. Many neuromuscular diseases affect muscle spindle function contributing, among others, to an unstable gait, frequent falls and ataxic behavior in the affected patients. Nevertheless, muscle spindles are usually ignored during examination and analysis of muscle function and when designing therapeutic strategies for neuromuscular diseases. This review summarizes the development and function of muscle spindles and the changes observed under pathological conditions, in particular in the various forms of muscular dystrophies.

Methocarbamol blocks muscular Nav 1.4 channels and decreases isometric force of mouse muscles.

BACKGROUND: The muscle relaxant methocarbamol is widely used for the treatment of muscle spasms and pain syndromes. To elucidate molecular mechanisms of its action, we studied its influence on neuromuscular transmission, on isometric muscle force, and on voltage-gated Na+ channels. METHODS: Neuromuscular transmission was investigated in murine diaphragm-phrenic nerve preparations and muscle force studied on mouse soleus muscles. Nav 1.4 channels and Nav 1.7 channels were functionally expressed in eukaryotic cell lines. RESULTS: Methocarbamol, at 2 mM, decreased the decay of endplate currents, slowed the decay of endplate potentials and reduced tetanic force of soleus muscles. The drug reversibly inhibited current flow through muscular Nav 1.4 channels, while neuronal Nav 1.7 channels were unaffected. CONCLUSIONS: The study provides evidence for peripheral actions of methocarbamol on skeletal muscle. Muscular Na+ channels are a molecular target of methocarbamol. Since Nav 1.7 currents were unaffected, methocarbamol is unlikely to exert its analgesic effect by directly blocking Nav 1.7 channels.

Impaired muscle spindle function in murine models of muscular dystrophy.

KEY POINTS: Muscular dystrophy patients suffer from progressive degeneration of skeletal muscle fibres, sudden spontaneous falls, balance problems, as well as gait and posture abnormalities. Dystrophin- and dysferlin-deficient mice, models for different types of muscular dystrophy with different aetiology and molecular basis, were characterized to investigate if muscle spindle structure and function are impaired. The number and morphology of muscle spindles were unaltered in both dystrophic mouse lines but muscle spindle resting discharge and their responses to stretch were altered. In dystrophin-deficient muscle spindles, the expression of the paralogue utrophin was substantially upregulated, potentially compensating for the dystrophin deficiency. The results suggest that muscle spindles might contribute to the motor problems observed in patients with muscular dystrophy. ABSTRACT: Muscular dystrophies comprise a heterogeneous group of hereditary diseases characterized by progressive degeneration of extrafusal muscle fibres as well as unstable gait and frequent falls. To investigate if muscle spindle function is impaired, we analysed their number, morphology and function in wildtype mice and in murine model systems for two distinct types of muscular dystrophy with very different disease aetiology, i.e. dystrophin- and dysferlin-deficient mice. The total number and the overall structure of muscle spindles in soleus muscles of both dystrophic mouse mutants appeared unchanged. Immunohistochemical analyses of wildtype muscle spindles revealed a concentration of dystrophin and ß-dystroglycan in intrafusal fibres outside the region of contact with the sensory neuron. While utrophin was absent from the central part of intrafusal fibres of wildtype mice, it was substantially upregulated in dystrophin-deficient mice. Single-unit extracellular recordings of sensory afferents from muscle spindles of the extensor digitorum longus muscle revealed that muscle spindles from both dystrophic mouse strains have an increased resting discharge and a higher action potential firing rate during sinusoidal vibrations, particularly at low frequencies. The response to ramp-and-hold stretches appeared unaltered compared to the respective wildtype mice. We observed no exacerbated functional changes in dystrophin and dysferlin double mutant mice compared to the single mutant animals. These results show alterations in muscle spindle afferent responses in both dystrophic mouse lines, which might cause an increased muscle tone, and might contribute to the unstable gait and frequent falls observed in patients with muscular dystrophy.

Alternative Splicing and the Intracellular Domain Mediate TM-agrin's Ability to Differentially Regulate the Density of Excitatory and Inhibitory Synapse-like Specializations in Developing CNS Neurons.

Agrin is a multi-domain protein best known for its essential function during formation of the neuromuscular junction. Alternative mRNA splicing at sites named y and z in the C-terminal part of agrin regulates its interaction with a receptor complex consisting of the agrin-binding low-density lipoprotein receptor-related protein 4 (Lrp4) and the muscle-specific kinase (MuSK). Isoforms with inserts at both splice sites bind to Lrp4, activate MuSK and are synaptogenic at the neuromuscular junction. Agrin is also expressed as a transmembrane protein in the central nervous system (CNS) but its function during interneuronal synapse formation is unclear. Recently we demonstrated that transfection of a full-length cDNA coding for transmembrane agrin (TM-agrin) in cultured embryonic cortical neurons induced an Lrp4-dependent but MuSK-independent increase in dendritic glutamatergic synapses and an Lrp4- and MuSK-independent reduction of inhibitory synapses. Here we show that presynaptic specializations were similarly affected by TM-agrin overexpression. In addition, we mapped the regions within TM-agrin responsible for TM-agrin's effects on dendritic aggregates of synapse-associated proteins. We show that the presence of a four amino acid insert at splice site y is essential for the increase in the density of puncta containing the postsynaptic density protein 95 kDa. This effect was independent of splice site z. The reduction of the gephyrin puncta density was independent of the entire extracellular part of TM-agrin but required a highly conserved serine residue in the intracellular domain of TM-agrin. These results provide further evidence for a function of TM-agrin during CNS synaptogenesis and demonstrate that different domains and alternative splicing of TM-agrin differentially affect excitatory and inhibitory synapse formation in cultured embryonic CNS neurons.

Acetylcholine receptors in the equatorial region of intrafusal muscle fibres modulate mouse muscle spindle sensitivity.

KEY POINTS: Acetylcholine receptors are aggregated in the central regions of intrafusal muscle fibres. Single unit muscle spindle afferent responses from isolated mouse extensor digitorum longus muscle were recorded in the absence of fusimotor input to ramp and hold stretches as well as to sinusoidal vibrations in the presence and absence of the acetylcholine receptor blockers d-tubocurarine and α-bungarotoxin. Proprioceptive afferent responses to both types of stretch were enhanced in the presence of either blocker. Blocking acetylcholine uptake and vesicular acetylcholine release by hemicholinium-3 also enhanced stretch-evoked responses. These results represent the first evidence that acetylcholine receptors negatively modulate muscle spindle responses to stretch. The data support the hypothesis that the sensory nerve terminal is able to release vesicles to fine-tune proprioceptive afferent sensitivity. ABSTRACT: Muscle spindles are complex stretch-sensitive mechanoreceptors. They consist of specialized skeletal muscle fibres, called intrafusal fibres, which are innervated in the central (equatorial) region by afferent sensory axons and in both polar regions by efferent γ-motoneurons. Previously it was shown that acetylcholine receptors (AChR) are concentrated in the equatorial region at the contact site between the sensory neuron and the intrafusal muscle fibre. To address the function of these AChRs, single unit sensory afferents were recorded from an isolated mouse extensor digitorum longus muscle in the absence of γ-motoneuron activity. Specifically, we investigated the responses of individual sensory neurons to ramp-and-hold stretches and sinusoidal vibrations before and after the addition of the competitive and non-competitive AChR blockers d-tubocurarine and α-bungarotoxin, respectively. The presence of either drug did not affect the resting action potential discharge frequency. However, the action potential frequencies in response to stretch were increased. In particular, frequencies of the dynamic peak and dynamic index to ramp-and-hold stretches were significantly higher in the presence of either drug. Treatment of muscle spindle afferents with the high-affinity choline transporter antagonist hemicholinium-3 similarly increased muscle spindle afferent firing frequencies during stretch. Moreover, the firing rate during sinusoidal vibration stimuli at low amplitudes was higher in the presence of α-bungarotoxin compared to control spindles also indicating an increased sensitivity to stretch. Collectively these data suggest a modulation of the muscle spindle afferent response to stretch by AChRs in the central region of intrafusal fibres possibly fine-tuning muscle spindle sensitivity.

The role of agrin, Lrp4 and MuSK during dendritic arborization and synaptogenesis in cultured embryonic CNS neurons.

The role of agrin, Lrp4 and MuSK, key organizers of neuromuscular synaptogenesis, in the developing CNS is only poorly understood. We investigated the role of these proteins in cultured mouse embryonic cortical neurons from wildtype and from Lrp4- and MuSK-deficient mice. Neurons from Lrp4-deficient mice had fewer but longer primary dendrites and a decreased density of puncta containing excitatory and inhibitory synapse-associated proteins. Neurons from MuSK-deficient mice had an altered dendritic branching pattern but no change in the density of puncta stained by antibodies against synapse-associated proteins. Transfection of TM-agrin compensated the dendritic branching deficits in Lrp4-deficient but not in MuSK-deficient neurons. TM-agrin transfection increased the density of excitatory synaptic puncta in MuSK-deficient but not in Lrp4-deficient mice and reduced the number of inhibitory synaptic puncta irrespective of MuSK and Lrp4 expression. Addition of purified soluble agrin to microisland cultures of cortical neurons revealed an Lrp4-dependent increase in the size and density of glutamatergic synaptic puncta and in mEPSC but not in mIPSC frequency and amplitude. Thus, agrin induced an Lrp4-independent increase in dendritic branch complexity, an Lrp4-dependent increase of excitatory synaptic puncta and an Lrp4- and MuSK-independent decrease in the density of puncta containing inhibitory synapse-associated proteins. These results establish selective roles for agrin, Lrp4 and MuSK during dendritogenesis and synaptogenesis in cultured CNS neurons.

Proprioception 2.0: novel functions for muscle spindles.

PURPOSE OF REVIEW: Muscle spindles are encapsulated mechanosensory receptors within skeletal muscle tissue that inform the central nervous system about the contractile status of each muscle. This information is required for any coordinated movement and for stable posture. This review summarizes recent findings regarding novel functions for proprioceptive feedback information, muscle spindle disease and the molecular basis of mechanoreception. RECENT FINDINGS: Muscle spindle function is not limited to regulating motor control but is also required for appropriate realignment of fractured bones, successful regeneration of spinal cord axons after injury and spinal alignment. Several proteins responsible for or modulating mechanotransduction in proprioceptive sensory neurons have been identified, including the Piezo2 channel as a candidate for the principal mechanotransduction channel. Many neuromuscular diseases are known to be accompanied by an impaired function of muscle spindles, resulting in a decline of motor performance and coordination in the patients. SUMMARY: Our knowledge regarding the molecular basis of muscle spindle function is still incomplete. However, increasing our understanding of mechanotransduction in muscle spindles is a prerequisite for finding appropriate strategies to prevent injuries due to unstable gait and frequent falls.

Neuronal LRP4 regulates synapse formation in the developing CNS.

The low-density lipoprotein receptor-related protein 4 (LRP4) is essential in muscle fibers for the establishment of the neuromuscular junction. Here, we show that LRP4 is also expressed by embryonic cortical and hippocampal neurons, and that downregulation of LRP4 in these neurons causes a reduction in density of synapses and number of primary dendrites. Accordingly, overexpression of LRP4 in cultured neurons had the opposite effect inducing more but shorter primary dendrites with an increased number of spines. Transsynaptic tracing mediated by rabies virus revealed a reduced number of neurons presynaptic to the cortical neurons in which LRP4 was knocked down. Moreover, neuron-specific knockdown of LRP4 by in utero electroporation of LRP4 miRNA in vivo also resulted in neurons with fewer primary dendrites and a lower density of spines in the developing cortex and hippocampus. Collectively, our results demonstrate an essential and novel role of neuronal LRP4 in dendritic development and synaptogenesis in the CNS.

A Cell Culture System to Investigate the Presynaptic Control of Subsynaptic Membrane Differentiation at the Neuromuscular Junction.

For decades the neuromuscular junction (NMJ) has been a favorite preparation to investigate basic mechanisms of synaptic function and development. As its function is to transmit action potentials in a 1:1 ratio from motor neurons to muscle fibers, the NMJ shows little or no functional plasticity, a property that makes it poorly suited to investigate mechanisms of use-dependent adaptations of synaptic function, which are thought to underlie learning and memory formation in the brain. On the other hand, the NMJ is unique in that the differentiation of the subsynaptic membrane is regulated by one major factor secreted from motor neurons, agrin. As a consequence, myotubes grown on a laminin substrate that is focally impregnated with recombinant neural agrin closely resemble the situation in vivo, where agrin secreted from motor neurons binds to the basal lamina of the NMJ's synaptic cleft to induce and maintain the subsynaptic muscle membrane. We provide here a detailed protocol through which acetylcholine receptor clusters are induced in cultured myotubes contacting laminin-attached agrin, enabling molecular, biochemical and cell biological analyses including high resolution microscopy in 4D. This preparation is ideally suited to investigate the mechanisms involved in the assembly of the postsynaptic muscle membrane, providing distinct advantages over inducing AChR clusters using soluble agrin.

Decrease in newly generated oligodendrocytes leads to motor dysfunctions and changed myelin structures that can be rescued by transplanted cells.

NG2-glia in the adult brain are known to proliferate and differentiate into mature and myelinating oligodendrocytes throughout lifetime. However, the role of these newly generated oligodendrocytes in the adult brain still remains little understood. Here we took advantage of the Sox10-iCreERT2 x CAG-eGFP x Esco2fl/fl mouse line in which we can specifically ablate proliferating NG2-glia in adult animals. Surprisingly, we observed that the generation of new oligodendrocytes in the adult brain was severely affected, although the number of NG2-glia remained stable due to the enhanced proliferation of non-recombined cells. This lack of oligodendrogenesis led to the elongation of the nodes of Ranvier as well as the associated paranodes, which could be locally rescued by myelinating oligodendrocytes differentiated from transplanted NG2-glia deriving from wildtype mice. Repetitive measurements of conduction velocity in the corpus callosum of awake animals revealed a progressive deceleration specifically in the mice lacking adult oligodendrogenesis that resulted in progressive motor deficits. In summary, here we demonstrated for the first time that axon function is not only controlled by the reliable organization of myelin, but also requires a dynamic and continuous generation of new oligodendrocytes in the adult brain. GLIA 2016;64:2201-2218.

Differential regulation of AChR clustering in the polar and equatorial region of murine muscle spindles.

Intrafusal fibers of muscle spindles are innervated in the central region by afferent sensory axons and at both polar regions by efferent γ-motoneurons. We previously demonstrated that both neuron-muscle contact sites contain cholinergic synapse-like specialisation, including aggregates of the nicotinic acetylcholine receptor (AChR). In this study we tested the hypothesis that agrin and its receptor complex (consisting of LRP4 and the tyrosine kinase MuSK) are involved in the aggregation of AChRs in muscle spindles, similar to their role at the neuromuscular junction. We show that agrin, MuSK and LRP4 are concentrated at the contact site between the intrafusal fibers and the sensory- and γ-motoneuron, respectively, and that they are expressed in the cell bodies of proprioceptive neurons in dorsal root ganglia. Moreover, agrin and LRP4, but not MuSK, are expressed in γ-motoneuron cell bodies in the ventral horn of the spinal cord. In agrin- and in MuSK-deficient mice, AChR aggregates are absent from the polar regions. In contrast, the subcellular concentration of AChRs in the central region where the sensory neuron contacts the intrafusal muscle fiber is apparently unaffected. Skeletal muscle-specific expression of miniagrin in agrin(-/-) mice in vivo is sufficient to restore the formation of γ-motoneuron endplates. These results show that agrin and MuSK are major determinants during the formation of γ-motoneuron endplates but appear dispensable for the aggregation of AChRs at the central region. Our results therefore suggest different molecular mechanisms for AChR clustering within two domains of intrafusal fibers.

The heparan sulfate proteoglycan agrin contributes to barrier properties of mouse brain endothelial cells by stabilizing adherens junctions.

Barrier characteristics of brain endothelial cells forming the blood-brain barrier (BBB) are tightly regulated by cellular and acellular components of the neurovascular unit. During embryogenesis, the accumulation of the heparan sulfate proteoglycan agrin in the basement membranes ensheathing brain vessels correlates with BBB maturation. In contrast, loss of agrin deposition in the vasculature of brain tumors is accompanied by the loss of endothelial junctional proteins. We therefore wondered whether agrin had a direct effect on the barrier characteristics of brain endothelial cells. Agrin increased junctional localization of vascular endothelial (VE)-cadherin, ß-catenin, and zonula occludens-1 (ZO-1) but not of claudin-5 and occludin in the brain endothelioma cell line bEnd5 without affecting the expression levels of these proteins. This was accompanied by an agrin-induced reduction of the paracellular permeability of bEnd5 monolayers. In vivo, the lack of agrin also led to reduced junctional localization of VE-cadherin in brain microvascular endothelial cells. Taken together, our data support the notion that agrin contributes to barrier characteristics of brain endothelium by stabilizing the adherens junction proteins VE-cadherin and ß-catenin and the junctional protein ZO-1 to brain endothelial junctions.

Formation of cholinergic synapse-like specializations at developing murine muscle spindles.

Muscle spindles are complex stretch-sensitive mechanoreceptors. They consist of specialized skeletal muscle fibers, called intrafusal fibers, which are innervated in the central (equatorial) region by afferent sensory axons and in both polar regions by efferent γ-motoneurons. We show that AChRs are concentrated at the γ-motoneuron endplate as well as in the equatorial region where they colocalize with the sensory nerve ending. In addition to the AChRs, the contact site between sensory nerve ending and intrafusal muscle fiber contains a high concentration of choline acetyltransferase, vesicular acetylcholine transporter and the AChR-associated protein rapsyn. Moreover, bassoon, a component of the presynaptic cytomatrix involved in synaptic vesicle exocytosis, is present in γ-motoneuron endplates but also in the sensory nerve terminal. Finally, we demonstrate that during postnatal development of the γ-motoneuron endplate, the AChR subunit stoichiometry changes from the γ-subunit-containing fetal AChRs to the ε-subunit-containing adult AChRs, similar and approximately in parallel to the postnatal subunit maturation at the neuromuscular junction. In contrast, despite the onset of ε-subunit expression during postnatal development the γ-subunit remains detectable in the equatorial region by subunit-specific antibodies as well as by analysis of muscle spindles from mice with genetically-labeled AChR γ-subunits. These results demonstrate an unusual maturation of the AChR subunit composition at the annulospiral endings and suggest that in addition to the recently described glutamatergic secretory system, the sensory nerve terminals are also specialized for cholinergic synaptic transmission, synaptic vesicle storage and exocytosis.

Anti-agrin autoantibodies in myasthenia gravis.

OBJECTIVE: Because the extracellular matrix protein agrin is essential for neuromuscular junction formation and maintenance, we tested the hypothesis that autoantibodies against agrin are present in sera from patients with myasthenia gravis (MG). METHODS: We determined the presence of anti-agrin antibodies in 54 sera from patients with generalized MG using a solid-phase ELISA with purified mini-agrin protein. Thirty of the 54 sera were seronegative for antibodies against the acetylcholine receptor (AChR) or muscle-specific tyrosine kinase (MuSK), 15 had elevated levels of anti-MuSK, and 9 had elevated levels of anti-AChR autoantibodies. Sixteen sera from healthy volunteers served as control. RESULTS: Five sera with elevated levels of anti-agrin antibodies were identified. The concentration of the antibodies ranged between 0.04 and 0.12 nM. Four of the 5 agrin-positive sera were also positive for anti-MuSK, one was positive for anti-AChR, and 2 had elevated levels of anti-low-density lipoprotein receptor-related protein 4 (LRP4) autoantibodies. Some of the sera stained adult mouse neuromuscular junctions and reacted with native mini-agrin expressed in 293HEK cells. CONCLUSIONS: The results provide evidence for agrin as a novel target protein for autoantibodies in patients with MG. Anti-agrin antibodies were always detected in combination with autoantibodies against MuSK, LRP4, or AChRs, indicating a high incidence of autoantibodies against several neuromuscular proteins in the agrin-positive MG cases.