Marco Morsch

Muscle specific kinase : organiser of synaptic membrane domains

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.

Pyridostigmine but not 3,4‐diaminopyridine exacerbates ACh receptor loss and myasthenia induced in mice by muscle‐specific kinase autoantibody

•u2002 A mouse model of anti‐muscle‐specific kinase (MuSK) myasthenia gravis was used to study the effect of pyridostigmine (a cholinesterase inhibitor drug commonly used in myasthenia) on the disease process at the neuromuscular junction. •u2002 In mice receiving injections of anti‐MuSK‐positive patient IgG, pyridostigmine treatment for 7–9 days did not prevent myasthenia, and even precipitated weakness. •u2002 Pyridostigmine treatment potentiated the anti‐MuSK‐induced reductions in postsynaptic acetylcholine receptor density and endplate potential (EPP) amplitude. •u2002 3,4‐Diaminopyridine, a drug that increases the number of quanta released (rather than the duration of each quantal response), elevated EPP amplitude without exacerbating the anti‐MuSK‐induced loss of acetylcholine receptors. •u2002 The results suggest that cholinergic‐ and MuSK‐mediated signalling may converge postsynaptically to regulate the mature acetylcholine receptor scaffold.

The established and emerging roles of astrocytes and microglia in amyotrophic lateral sclerosis and frontotemporal dementia

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two progressive, fatal neurodegenerative syndromes with considerable clinical, genetic and pathological overlap. Clinical symptoms of FTD can be seen in ALS patients and vice versa. Recent genetic discoveries conclusively link the two diseases, and several common molecular players have been identified (TDP-43, FUS, C9ORF72). The definitive etiologies of ALS and FTD are currently unknown and both disorders lack a cure. Glia, specifically astrocytes and microglia are heavily implicated in the onset and progression of neurodegeneration witnessed in ALS and FTD. In this review, we summarize the current understanding of the role of microglia and astrocytes involved in ALS and FTD, highlighting their recent implications in neuroinflammation, alterations in waste clearance involving phagocytosis and the newly described glymphatic system, and vascular abnormalities. Elucidating the precise mechanisms of how astrocytes and microglia are involved in ALS and FTD will be crucial in characterizing these two disorders and may represent more effective interventions for disease progression and treatment options in the future.

Sequence of Age-Associated Changes to the Mouse Neuromuscular Junction and the Protective Effects of Voluntary Exercise

Loss of connections between motor neurons and skeletal muscle fibers contribute to motor impairment in old age, but the sequence of age-associated changes that precede loss of the neuromuscular synapse remains uncertain. Here we determine changes in the size of neuromuscular synapses within the tibialis anterior muscle across the life span of C57BL/6J mice. Immunofluorescence, confocal microscopy and morphometry were used to measure the area occupied by nerve terminal synaptophysin staining and postsynaptic acetylcholine receptors at motor endplates of 2, 14, 19, 22, 25 and 28month old mice. The key findings were: 1) At middle age (14-months) endplate acetylcholine receptors occupied 238±11 µm2 and nerve terminal synaptophysin 168±14 µm2 (mean ± SEM). 2) Between 14-months and 19-months (onset of old age) the area occupied by postsynaptic acetylcholine receptors declined 30%. At many endplates the large acetylcholine receptor plaque became fragmented into multiple smaller acetylcholine receptor clusters. 3) Between 19- and 25-months, the fraction of endplate acetylcholine receptors covered by synaptophysin fell 21%. By 28-months, half of the endplates imaged retained ≤50 µm2 area of synaptophysin staining. 4) Within aged muscles, the degree to which an endplate remained covered by synaptophysin did not depend upon the total area of acetylcholine receptors, nor upon the number of discrete receptor clusters. 5) Voluntary wheel-running exercise, beginning late in middle-age, prevented much of the age-associated loss of nerve terminal synaptophysin. In summary, a decline in the area of endplate acetylcholine receptor clusters at the onset of old age was followed by loss of nerve terminal synaptophysin from the endplate. Voluntary running exercise, begun late in middle age, substantially inhibited the loss of nerve terminal from aging motor endplates.

Muscle specific kinase autoantibodies cause synaptic failure through progressive wastage of postsynaptic acetylcholine receptors.

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 15 days. 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.

Modulation of the Ca2+ conductance of nicotinic acetylcholine receptors by Lypd6.

The agonist binding sensitivity and desensitization kinetics of nicotinic acetylcholine receptors (nAChRs) can be modulated by snake venom neurotoxins and related endogenous small proteins of the uPAR-Ly6 family. Here we identify Lypd6, a distantly related member of the u-PAR/Ly-6 family expressed in neurons as a novel modulator of nAChRs. Lypd6 overexpressed in trigeminal ganglia neurons selectively enhanced the Ca2+-component of nicotine-evoked currents through nAChRs, as evidenced by comparative whole-cell patch clamp recordings and Ca2+-imaging in wildtype and transgenic mice overexpressing Lypd6. In contrast, a knockdown of Lypd6 expression using siRNAs selectively reduced nicotine-evoked Ca2+-currents. Pharmacological experiments revealed that the nAChRs involved in this process are heteromers. Transgenic mice displayed behaviors that were indicative of an enhanced cholinergic tone, such as a higher locomotor arousal, increased prepulse-inhibition and hypoalgesia. These mice overexpressing Lypd6 mice were also more sensitive to the analgesic effects of nicotine. Transgenic mice expressing siRNAs directed against Lypd6 were unable to procreate, thus indicating a vital role for this protein. Taken together, Lypd6 seems to constitute a novel modulator of nAChRs that affects receptor function by selectively increasing Ca2+-influx through this ion channels.

Effects of the ß2-adrenoceptor agonist, albuterol, in a mouse model of anti-MuSK myasthenia gravis.

The β2-adrenergic receptor agonist, albuterol, has been reported beneficial in treating several forms of congenital myasthenia. Here, for the first time, we examined the potential benefit of albuterol in a mouse model of anti-Muscle Specific Kinase (MuSK) myasthenia gravis. Mice received 15 daily injections of IgG from anti-MuSK positive patients, which resulted in whole-body weakness. At neuromuscular junctions in the tibialis anterior and diaphragm muscles the autoantibodies caused loss of postsynaptic acetylcholine receptors, and reduced the amplitudes of the endplate potential and spontaneous miniature endplate potential in the diaphragm muscle. Treatment with albuterol (8 mg/kg/day) during the two-week anti-MuSK injection series reduced the degree of weakness and weight loss, compared to vehicle-treated mice. However, the compound muscle action potential recorded from the gastrocnemius muscle displayed a decremental response in anti-MuSK-injected mice whether treated with albuterol or vehicle. Ongoing albuterol treatment did not increase endplate potential amplitudes compared to vehicle-treated mice nor did it prevent the loss of acetylcholine receptors from motor endplates. On the other hand, albuterol treatment significantly reduced the degree of fragmentation of endplate acetylcholine receptor clusters and increased the extent to which the remaining receptor clusters were covered by synaptophysin-stained nerve terminals. The results provide the first evidence that short-term albuterol treatment can ameliorate weakness in a robust mouse model of anti-MuSK myasthenia gravis. The results also demonstrate that it is possible for albuterol treatment to reduce whole-body weakness without necessarily reversing myasthenic impairment to the structure and function of the neuromuscular junction.

In vivo characterization of microglial engulfment of dying neurons in the zebrafish spinal cord

Microglia are specialized phagocytes in the vertebrate central nervous system (CNS). As the resident immune cells of the CNS they play an important role in the removal of dying neurons during both development and in several neuronal pathologies. Microglia have been shown to prevent the diffusion of damaging degradation products of dying neurons by engulfment and ingestion. Here we describe a live imaging approach that uses UV laser ablation to selectively stress and kill spinal neurons and visualize the clearance of neuronal remnants by microglia in the zebrafish spinal cord. In vivo imaging confirmed the motile nature of microglia within the uninjured spinal cord. However, selective neuronal ablation triggered rapid activation of microglia, leading to phagocytic uptake of neuronal debris by microglia within 20–30 min. This process of microglial engulfment is highly dynamic, involving the extension of processes toward the lesion site and consequently the ingestion of the dying neuron. 3D rendering analysis of time-lapse recordings revealed the formation of phagosome-like structures in the activated microglia located at the site of neuronal ablation. This real-time representation of microglial phagocytosis in the living zebrafish spinal cord provides novel opportunities to study the mechanisms of microglia-mediated neuronal clearance.

Electrophysiological analysis of neuromuscular synaptic function in myasthenia gravis patients and animal models

Study of the electrophysiological function of the neuromuscular junction (NMJ) is instrumental in the understanding of the symptoms and pathophysiology of myasthenia gravis (MG), an autoimmune disorder characterized by fluctuating and fatigable muscle weakness. Most patients have autoantibodies to the acetylcholine receptor at the NMJ. However, in recent years autoantibodies to other crucial postsynaptic membrane proteins have been found in previously seronegative MG patients. Electromyographical recording of compound and single-fibre muscle action potentials provides a crucial in vivo method to determine neuromuscular transmission failure while ex vivo (miniature) endplate potential recordings can reveal the precise synaptic impairment. Here we will review these electrophysiological methods used to assess NMJ function and discuss their application and typical results found in the diagnostic and experimental study of patients and animal models of the several forms of MG.

Muscle-specific kinase (MuSK) autoantibodies suppress the MuSK pathway and ACh receptor retention at the mouse neuromuscular junction.

Myasthenic anti‐muscle‐specific‐kinase (MuSK) IgG was injected into mice to study its effect upon the MuSK signalling pathway and the homeostasis of postsynaptic acetylcholine receptor packing at the neuromuscular junction. Densities of MuSK, activated Src kinase, phosphorylated ACh receptors and rapsyn were all reduced at motor endplates while β‐dystroglycan was unaffected. Pulse‐labelling showed that the slow decline in junctional ACh receptor density could be explained largely by diminished retention of ACh receptors within the postsynaptic membrane scaffold. The results suggest that anti‐MuSK IgG reduces the density of MuSK, associated tyrosine phosphorylation and retention of junctional ACh receptors within the postsynaptic membrane.