Mark M. Rich

Rescue of the spinal muscular atrophy phenotype in a mouse model by early postnatal delivery of SMN

Spinal muscular atrophy (SMA), the most common autosomal recessive neurodegenerative disease affecting children, results in impaired motor neuron function. Despite knowledge of the pathogenic role of decreased survival motor neuron (SMN) protein levels, efforts to increase SMN have not resulted in a treatment for patients. We recently demonstrated that self-complementary adeno-associated virus 9 (scAAV9) can infect ∼60% of motor neurons when injected intravenously into neonatal mice. Here we use scAAV9-mediated postnatal day 1 vascular gene delivery to replace SMN in SMA pups and rescue motor function, neuromuscular physiology and life span. Treatment on postnatal day 5 results in partial correction, whereas postnatal day 10 treatment has little effect, suggesting a developmental period in which scAAV9 therapy has maximal benefit. Notably, we also show extensive scAAV9-mediated motor neuron transduction after injection into a newborn cynomolgus macaque. This demonstration that scAAV9 traverses the blood-brain barrier in a nonhuman primate emphasizes the clinical potential of scAAV9 gene therapy for SMA.

Mice lacking the myotonic dystrophy protein kinase develop a late onset progressive myopathy.

Myotonic dystrophy (DM) is an autosomal dominant disorder resulting from the expansion of a CTG repeat in the 3′ untranslated region of a putative protein kinase (DMPK). To elucidate the role of DMPK in DM pathogenesis we have developed DMPK deficient (DMPK−/−) mice. DMPK−/− mice develop a late-onset, progressive skeletal myopathy that shares some pathological features with DM. Muscles from mature mice show variation in fibre size, increased fibre degeneration and fibrosis. Adult DMPK−/− mice show ultrastructural changes in muscle and a 50% decrease in force generation compared to young mice. Our results indicate that DMPK may be necessary for the maintenance of skeletal muscle structure and function and suggest that a decrease in DMPK levels may contribute to DM pathology.

Impaired Synaptic Vesicle Release and Immaturity of Neuromuscular Junctions in Spinal Muscular Atrophy Mice

The motor neuron disease spinal muscular atrophy (SMA) causes profound muscle weakness that most often leads to early death. At autopsy, SMA is characterized by loss of motor neurons and muscle atrophy, but the initial cellular events that precipitate motor unit dysfunction and loss remain poorly characterized. Here, we examined the function and corresponding structure of neuromuscular junction (NMJ) synapses in a mouse model of severe SMA (hSMN2/delta7SMN/mSmn−/−). Surprisingly, most SMA NMJs remained innervated even late in the disease course; however they showed abnormal synaptic transmission. There was a two-fold reduction in the amplitudes of the evoked endplate currents (EPCs), but normal spontaneous miniature EPC (MEPC) amplitudes. These features in combination indicate reduced quantal content. SMA NMJs also demonstrated increased facilitation suggesting a reduced probability of vesicle release. By electron microscopy, we found a decreased density of synaptic vesicles that is likely to contribute to the reduced release probability. In addition to presynaptic defects, there were postsynaptic abnormalities. EPC and MEPC decay time constants were prolonged because of a slowed switch from the fetal acetylcholine receptor (AChR) γ-subunit to the adult ε-subunit. There was also reduced size of AChR clusters and small myofibers, which expressed an immature pattern of myosin heavy chains. Together these results indicate that impaired synaptic vesicle release at NMJs in severe SMA is likely to contribute to failed postnatal maturation of motor units and muscle weakness.

Direct muscle stimulation in acute quadriplegic myopathy

We have previously found that muscle is electrically inexcitable in severe acute quadriplegic myopathy (AQM). In contrast, muscle retains normal electrical excitability in peripheral neuropathy. To study the relationship between muscle electrical excitability and all types of flaccid weakness occurring in the intensive care unit, we identified 14 critically ill, weak patients and measured the amplitude of compound muscle action potentials (CMAPs) obtained with direct muscle stimulation (dmCMAP) and with nerve stimulation (neCMAP). In 11 of 14 patients dmCMAP amplitudes were reduced and the ratio of the neCMAP amplitude to the dmCMAP amplitude (nerve/muscle ratio) was indicative of loss of muscle electrical excitability. In 2 other patients, the nerve/muscle ratio indicated neuropathy. Direct muscle stimulation may allow differentiation of AQM from neuropathy even in comatose or encephalopathic critically ill patients. AQM may be more common than has previously been appreciated.

Muscle is electrically inexcitable in acute quadriplegic myopathy

We directly stimulated muscle in three patients with acute quadriplegic myopathy to determine whether paralyzed muscle in this syndrome is electrically excitable. Two of the patients had been treated with neuromuscular blocking agents and corticosteroids, and one patient had been treated with corticosteroids alone. We found that paralyzed muscle is electrically inexcitable in affected patients. Muscle regained electrical excitability over weeks to months. The recovery of muscle excitability paralleled the clinical recovery of patients, suggesting that paralysis in this syndrome is secondary to electrical inexcitability of muscle membrane.

Disruption of TrkB-Mediated Signaling Induces Disassembly of Postsynaptic Receptor Clusters at Neuromuscular Junctions

Neurotrophins and tyrosine receptor kinase (Trk) receptors are expressed in skeletal muscle, but it is unclear what functional role Trk-mediated signaling plays during postnatal life. Full-length TrkB (trkB.FL) as well as truncated TrkB (trkB.t1) were found to be localized primarily to the postsynaptic acetylcholine receptor- (AChR-) rich membrane at neuromuscular junctions. In vivo, dominant-negative manipulation of TrkB signaling using adenovirus to overexpress trkB.t1 in mouse sternomastoid muscle fibers resulted in the disassembly of postsynaptic AChR clusters at neuromuscular junctions, similar to that observed in mutant trkB+/- mice. When TrkB-mediated signaling was disrupted in cultured myotubes in the absence of motor nerve terminals and Schwann cells, agrin-induced AChR clusters were also disassembled. These results demonstrate a novel role for neurotrophin signaling through TrkB receptors on muscle fibers in the ongoing maintenance of postsynaptic AChR regions.

Early development of critical illness myopathy and neuropathy in patients with severe sepsis

Objectives: To characterize the prevalence, time of onset, and cause of neuromuscular dysfunction in patients with severe sepsis. Methods: We conducted a prospective cohort study in which participants with severe sepsis underwent weekly neurologic examinations and nerve conduction studies (NCSs) within 72 hours of developing severe sepsis until intensive care unit (ICU) discharge. Electromyography was preformed if clinical weakness developed or if there was a significant reduction in nerve conduction response amplitudes. Results: Abnormal NCS were present upon enrollment in 63% of patients (31/48). The presence of abnormal baseline NCS was predictive of hospital mortality (55% vs 0% for patients with normal baseline NCS; p < 0.001). Development of acquired neuromuscular dysfunction could be predicted by NCS done on day 7. Twenty patients remained in the ICU long enough to have serial NCSs; 50% of these patients developed acquired neuromuscular dysfunction. Most patients with acquired neuromuscular dysfunction had electrophysiologic evidence of both critical illness myopathy and critical illness neuropathy. Conclusion: Changes in nerve conduction studies occur in the majority of patients early in the course of severe sepsis and predict the development of acquired neuromuscular dysfunction and mortality in intensive care unit patients. Most patients with acquired neuromuscular dysfunction after sepsis have both critical illness myopathy and critical illness neuropathy.

Crucial Role of Sodium Channel Fast Inactivation in Muscle Fibre Inexcitability in a Rat Model of Critical Illness Myopathy

Critical illness myopathy is an acquired disorder in which skeletal muscle becomes electrically inexcitable. We previously demonstrated that inactivation of Na+ channels contributes to inexcitability of affected fibres in an animal model of critical illness myopathy in which denervated rat skeletal muscle is treated with corticosteroids (steroid denervated; SD). Our previous work, however, did not address the relative importance of membrane depolarization versus a shift in the voltage dependence of fast inactivation in causing inexcitability. It also remained unknown whether changes in the voltage dependence of activation or slow inactivation play a role in inexcitability. In the current study we found that a hyperpolarizing shift in the voltage dependence of fast inactivation of Na+ channels is the principal factor underlying inexcitability in SD fibres. Although depolarization tends to decrease excitability, it is insufficient to account for inexcitability in SD fibres since many normal and denervated fibres retain normal excitability when depolarized to the same resting potentials as affected SD fibres. Changes in the voltage dependence of activation and slow inactivation of Na+ channels were also observed in SD fibres; however, the changes appear to increase rather than decrease excitability. These results highlight the importance of the change in fast inactivation in causing inexcitability of SD fibres.

Survival Motor Neuron Protein in Motor Neurons Determines Synaptic Integrity in Spinal Muscular Atrophy

The inherited motor neuron disease spinal muscular atrophy (SMA) is caused by deficient expression of survival motor neuron (SMN) protein and results in severe muscle weakness. In SMA mice, synaptic dysfunction of both neuromuscular junctions (NMJs) and central sensorimotor synapses precedes motor neuron cell death. To address whether this synaptic dysfunction is due to SMN deficiency in motor neurons, muscle, or both, we generated three lines of conditional SMA mice with tissue-specific increases in SMN expression. All three lines of mice showed increased survival, weights, and improved motor behavior. While increased SMN expression in motor neurons prevented synaptic dysfunction at the NMJ and restored motor neuron somal synapses, increased SMN expression in muscle did not affect synaptic function although it did improve myofiber size. Together these data indicate that both peripheral and central synaptic integrity are dependent on motor neurons in SMA, but SMN may have variable roles in the maintenance of these different synapses. At the NMJ, it functions at the presynaptic terminal in a cell-autonomous fashion, but may be necessary for retrograde trophic signaling to presynaptic inputs onto motor neurons. Importantly, SMN also appears to function in muscle growth and/or maintenance independent of motor neurons. Our data suggest that SMN plays distinct roles in muscle, NMJs, and motor neuron somal synapses and that restored function of SMN at all three sites will be necessary for full recovery of muscle power.

Poliomyelitis due to West Nile virus.

To the Editor: Poliomyelitis is a clinical syndrome defined by the presence of fever, meningitis, and flaccid paralysis. In the United States, this syndrome was historically associated with infecti...