Linda L. Kusner

Molecular architecture of the neuromuscular junction

The neuromuscular junction (NMJ) is a complex structure that serves to efficiently communicate the electrical impulse from the motor neuron to the skeletal muscle to signal contraction. Over the last 200 years, technological advances in microscopy allowed visualization of the existence of a gap between the motor neuron and skeletal muscle that necessitated the existence of a messenger, which proved to be acetylcholine. Ultrastructural analysis identified vesicles in the presynaptic nerve terminal, which provided a beautiful structural correlate for the quantal nature of neuromuscular transmission, and the imaging of synaptic folds on the muscle surface demonstrated that specializations of the underlying protein scaffold were required. Molecular analysis in the last 20 years has confirmed the preferential expression of synaptic proteins, which is guided by a precise developmental program and maintained by signals from nerve. Although often overlooked, the Schwann cell that caps the NMJ and the basal lamina is proving to be critical in maintenance of the junction. Genetic and autoimmune disorders are known that compromise neuromuscular transmission and provide further insights into the complexities of NMJ function as well as the subtle differences that exist among NMJ that may underlie the differential susceptibility of muscle groups to neuromuscular transmission diseases. In this review we summarize the synaptic physiology, architecture, and variations in synaptic structure among muscle types. The important roles of specific signaling pathways involved in NMJ development and acetylcholine receptor (AChR) clustering are reviewed. Finally, genetic and autoimmune disorders and their effects on NMJ architecture and neuromuscular transmission are examined. Muscle Nerve, 2005

Nitric oxide synthase is concentrated at the skeletal muscle endplate.

NO performs a wide array of cell signaling functions. Neuronal NO synthase (nNOS) immunoreactivity and nicotinamide adenine dinucleotide phosphate diaphorase (NDP) activity, a marker of nNOS, were concentrated at adult rat neuromuscular junctions and persisted in denervated muscle indicating the localization of the enzyme to the postsynaptic surface. The concentration of nNOS at the muscle endplate suggests NO could serve as a messenger pre- and postsynapticly.

Constitutive properties, not molecular adaptations, mediate extraocular muscle sparing in dystrophic mdx mice

Extraocular muscle (EOM) is spared in Duchenne muscular dystrophy. Here, we tested putative EOM sparing mechanisms predicted from existing dystrophinopathy models. Data show that mdx mouse EOM contains dystrophin‐glycoprotein complex (DGC)‐competent and DGC‐deficient myofibers distributed in a fiber type‐specific pattern. Up‐regulation of a dystrophin homologue, utrophin, mediates selective DGC retention. Counter to the DGC mechanical hypothesis, an intact DGC is not a precondition for EOM sarcolemmal integrity, and active adaptation at the level of calcium homeostasis is not mechanistic in protection. A partial, fiber type‐specific retention of antiischemic nitric oxide to vascular smooth muscle signaling is not a factor in EOM sparing, because mice deficient in dystrophin and α‐syntrophin, which localizes neuronal nitric oxide synthase to the sarcolemma, have normal EOMs. Moreover, an alternative transmembrane protein, α7β1 integrin, does not appear to substitute for the DGC in EOM. Finally, genomewide expression profiling showed that EOM does not actively adapt to dystrophinopathy but identified candidate genes for the constitutive protection of mdx EOM. Taken together, data emphasize the conditional nature of dystrophinopathy and the potential importance of nonmechanical DGC roles and support the hypothesis that broad, constitutive structural cell signaling, and/or biochemical differences between EOM and other skeletal muscles are determinants of differential disease responsiveness.

Deficiency of decay accelerating factor and CD59 leads to crisis in experimental myasthenia.

In myasthenia gravis (MG), neuromuscular transmission is disrupted due to the production of autoantibodies against acetylcholine receptors (AChR). In previous work, we showed that decay accelerating factor (DAF or CD55), an intrinsic cell surface complement regulator that disables C3/C5 amplification convertases, protects against receptor loss and muscle weakness. In this study, we examined whether, and if so, to what extent CD59, a downstream intrinsic cell surface regulator that prevents assembly of membrane attack complexes (MACs), contributes to this protection. Twenty-four hours after anti-AChR injection, we found that CD59a-/- mice did not significantly differ from WTs, all Daf1-/- CD59a-/- mice either died or required euthanasia. At 48h, Daf1-/- were significantly weaker than CD59a-/- and WT mice, and for these mice immunohistochemistry revealed marked C9 deposition at postsynaptic junctions, radioimmunoassays showed reductions in AChR levels, and electron microscopy demonstrated massive junctional damage. These data indicate that DAF serves as the initial shield that protects the neuromuscular junction whereas CD59 is a further barrier. They argue that complement inhibitor, particularly if targeted to the receptor, could then have therapeutic value in human MG.

Ocular myasthenia: diagnosis, treatment, and pathogenesis.

Background:Although myasthenia gravis (MG) is often considered the best-understood autoimmune disorder and effective treatments have controlled life-threatening complications, the pathogenesis of ocular myasthenia (OM) remains enigmatic, and its clinical consequences offer therapeutic challenges. Review Summary:About half of patients with MG present with visual complaints of droopy eyelids or double vision, and many will remain with purely ocular muscle weakness without generalized weakness, defined as OM. OM may be confused with disorders of the brainstem, ocular motor nerves, and eye muscles. Frustrating for the clinician, confirmatory tests such as the edrophonium test, serum acetylcholine receptor antibodies, and standard electrodiagnostic evaluations may fail to positively identify the clinical suspicion of OM. Patients may derive relief from nonpharmacologic interventions and cholinesterase inhibitors, but most will desire better symptom control with corticosteroids or need other immunosuppression. Early corticosteroid therapy may reduce the probability of generalization of the disease. The reasons for ocular muscle involvement by OM include physiologic and cellular properties of the ocular motor system and the unique immunology of OM, which, when better understood, will lead to novel treatments. Conclusions:OM is a challenging disorder for the clinician and scientist, with both learning from the other for the betterment of the patient. The future requires answers to why the ocular muscles are so frequently involved by MG, whether the generalization of the disease may be limited by early corticosteroid treatment, and what treatment options may be developed which will improve symptoms without long-term complications.

Nitric oxide synthase in aging rat skeletal muscle.

The neuronal isoform of nitric oxide synthase (NOS) is expressed at high concentrations in skeletal muscle, and NO influences muscle contractility, glucose utilization, and free radical damage or protection. NOS activity and expression was evaluated in extensor digitorum longus (EDL), soleus, and diaphragm of 8 and 24 month old Fisher 344 rats. In 8-month-old animals, NOS activity was highest in EDL, which contained the highest percentage of NOS containing fibers, and was lowest in soleus. NOS activity and percentage of NOS containing fibers was significantly reduced in all muscle groups with age. To determine if NOS reduction correlated with free radical injury the level of lipid peroxidation, as measured by malonaldehyde equivalents, was determined. With age lipid peroxidation increased in EDL, was reduced in diaphragm, and showed a non-significant change in soleus. Therefore, a straightforward reduction of NOS activity does not correlate with lipid peroxidation. The reduction of NOS with age in skeletal muscle may be most significant for muscle metabolism and force production and be of limited significance for free radical metabolism.

The gamma-Subunit of the Acetylcholine Receptor Is Not Expressed in the Levator Palpebrae Superioris

Article abstract-In extraocular muscle (EOM), expression of the gamma-subunit, which is associated with the fetal-type acetylcholine receptor (AChR), may offer a differential target for immune-mediated damage and could explain the preponderance of ocular manifestations caused by myasthenia gravis (MG). Using Poly(A) sup + RNA hybridization, we investigated expression of the gamma-subunit in bovine levator palpebrae superioris (LP), a muscle also differentially involved by MG. There were no transcripts of the gamma-subunit of the AChR, but the epsilon-subunit, associated with the adult-type AChR, was present. The results indicate that the susceptibility of LP to MG is not mediated by gamma-subunit expression and suggest that multiterminal fibers in EOM may be the site of gamma-subunit expression. NEUROLOGY 1995;45: 516-518

Ryanodine receptor gene expression thymomas.

Ryanodine receptor (RyR) antibodies are present in sera of myasthenics with thymoma, and their titer correlates with morbidity and mortality. We investigated whether skeletal muscle RyR expression in thymic tissues could be the source of immune sensitization to the RyR. Skeletal muscle RyR gene expression was investigated using reverse transcription followed by semiquantitative polymerase chain reaction. Hyperplastic and normal thymuses expressed significant levels of RyR, but RyR gene transcripts was statistically less likely in thymomas than in hyperplastic and normal thymus (P < 0.05). The presence of RyR transcripts in thymomas did not correlate with myasthenic manifestations, thymic pathology, or serum RyR antibodies. We conclude that the skeletal muscle RyR in thymoma is not the inciting antigen for immune sensitization to RyR epitopes in thymoma‐associated myasthenia gravis.

Heme oxygenase-2 expression at rat neuromuscular junctions

The neuromuscular junction is specialized for rapid transmission of electrical signals. Nitric oxide synthase (NOS) is concentrated at the junction, and NO modulates transmission and could influence signaling pathways. Increasing evidence suggests that carbon monoxide (CO) serves as a neurotransmitter, and heme oxygenase (HO), the enzyme that catalyzes the formation of CO, is often colocalized with NOS. Immunoreactivity for HO-2 was present at rat neuromuscular junctions of leg muscles and persisted in denervated muscle indicating the localization of the enzyme to the postsynaptic surface. In contrast, HO-2 immunoreactivity was absent from the en grappe and orbital en plaque endplates of extraocular muscle (EOM), while only the global en plaque endplates possessed HO-2 immunoreactivity. The difference between EOM and leg endplates may arise from EOMs unique physiology. The presence of HO-2 at neuromuscular junctions suggests CO could serve as a pre- and post-synaptic messenger.

MicroRNA and mRNA expression associated with ectopic germinal centers in thymus of myasthenia gravis

Background A characteristic pathology of early onset myasthenia gravis is thymic hyperplasia with ectopic germinal centers (GC). However, the mechanisms that trigger and maintain thymic hyperplasia are poorly characterized. Dysregulation of small, non-coding microRNAs (miRNAs) and their target genes has been identified in the pathology of several autoimmune diseases. We assessed the miRNA and mRNA profiles of the MG thymus and have investigated their role in GC formation and maintenance. Methods MG thymus samples were assessed by histology and grouped based upon the appearance of GC; GC positive and GC negative. A systems biology approach was used to study the differences between the groups. Our study included miRNA and mRNA profiling, quantitative real-time PCR validation, miRNA target identification, pathway analysis, miRNA-mRNA reciprocal expression pairing and interaction. Results Thirty-eight mature miRNAs and forty-six annotated mRNA transcripts were differentially expressed between the two groups (>1.5 fold change, ANOVA p<0.05). The miRNAs were found to be involved in immune response pathways and identified in other autoimmune diseases. The cellular and molecular functions of the mRNAs showed involvement in cell death and cell survival, cellular proliferation, cytokine signaling and extra-cellular matrix reorganization. Eleven miRNA and mRNA pairs were reciprocally regulated. The Regulator of G protein Signalling 13 (RGS13), known to be involved in GC regulation, was identified in specimens with GC and was paired with downregulation of miR-452-5p and miR-139-5p. MiRNA target sites were validated by dual luciferase assay. Transfection of miRNA mimics led to down regulation of RGS13 expression in Raji cells. Conclusion Our study indicates a distinct miRNA and mRNA expression pattern in ectopic GC in MG thymus. These miRNAs and mRNAs are involved in regulatory pathways common to inflammation and immune response, cell cycle regulation and anti-apoptotic pathways suggesting their involvement in support of GC formation in the thymus. We demonstrate for the first time that miR-139-5p and miR-452-5p negatively regulate RGS13 expression.