Acetylcholine receptor antibodies in a patient with sensory neuropathy.

Antibodies to acetylcholine receptor (AChR) are detected in the vast majority of patients with generalized myasthenia gravis (MG) and are rarely detected in significant titer in other autoimmune diseases. We report a patient with an axonal predominately sensory neuropathy for over 12 years with persistent binding and modulating AChR antibodies as well as striational muscle antibodies with no evidence of MG or any neoplastic disease.

Functional α6ß4 acetylcholine receptor expression enables pharmacological testing of nicotinic agonists with analgesic properties.

The α6ß4 nicotinic acetylcholine receptor (nAChR) is enriched in dorsal root ganglia neurons and is an attractive non-opioid therapeutic target for pain. However, difficulty expressing human α6ß4 receptors in recombinant systems has precluded drug discovery. Here, genome-wide screening identified accessory proteins that enable reconstitution of human α6ß4 nAChRs. BARP, an auxiliary subunit of voltage-dependent calcium channels, promoted α6ß4 surface expression while IRE1α, an unfolded protein response sensor, enhanced α6ß4 receptor assembly. Effects on α6ß4 involve BARP's N-terminal region and IRE1α's splicing of XBP1 mRNA. Furthermore, clinical efficacy of nicotinic agents in relieving neuropathic pain best correlated with their activity on α6ß4. Finally, BARP-knockout, but not NACHO-knockout mice lacked nicotine-induced antiallodynia, highlighting the functional importance of α6ß4 in pain. These results identify roles for IRE1α and BARP in neurotransmitter receptor assembly and unlock drug discovery for the previously elusive α6ß4 receptor.

Phosphorylation of α-dystrobrevin is essential for αkap accumulation and acetylcholine receptor stability.

The maintenance of a high density of the acetylcholine receptor (AChR) is the hallmark of the neuromuscular junction. Muscle-specific anchoring protein (αkap) encoded within the calcium/calmodulin-dependent protein kinase IIα (CAMK2A) gene is essential for the maintenance of AChR clusters both in vivo and in cultured muscle cells. The underlying mechanism by which αkap is maintained and regulated remains unknown. Here, using human cell lines, fluorescence microscopy, and pulldown and immunoblotting assays, we show that α-dystrobrevin (α-dbn), an intracellular component of the dystrophin glycoprotein complex, directly and robustly promotes the stability of αkap in a concentration-dependent manner. Mechanistically, we found that the phosphorylatable tyrosine residues of α-dbn are essential for the stability of α-dbn itself and its interaction with αkap, with substitution of three tyrosine residues in the α-dbn C terminus with phenylalanine compromising the αkap-α-dbn interaction and significantly reducing both αkap and α-dbn accumulation. Moreover, the αkap-α-dbn interaction was critical for αkap accumulation and stability. We also found that the absence of either αkap or α-dbn markedly reduces AChRα accumulation and that overexpression of α-dbn or αkap in cultured muscle cells promotes the formation of large agrin-induced AChR clusters. Collectively, these results indicate that the stability of αkap and α-dbn complex plays an important role in the maintenance of high-level expression of AChRs.

Stimulation of Acetylcholine Release and Pharmacological Potentiation of Cholinergic Transmission Affect Cholinergic Receptor Expression Differently during Visual Conditioning.

Cholinergic stimulation coupled with visual conditioning enhances the visual acuity and cortical responses in the primary visual cortex. To determine which cholinergic receptors are involved in these processes, qRT-PCR was used. Two modes of cholinergic enhancement were tested: a phasic increase of acetylcholine release by an electrical stimulation of the basal forebrain cholinergic nucleus projecting to the visual cortex, or a tonic pharmacological potentiation of the cholinergic transmission by the acetylcholine esterase inhibitor, donepezil. A daily visual exposure to sine-wave gratings (training) was paired with the cholinergic enhancement, up to 14 days. qRT-PCR was performed at rest, 10 min, one week or two weeks of visual/cholinergic training with samples of the visual and somatosensory cortices, and the BF for determining mRNA expression of muscarinic receptor subtypes (m1, m2, m3, m4, m5), nicotinic receptor subunits (α3, α4, α7, ß2, ß4), and NMDA receptors, GAD65 and ChAT, as indexes of cortical plasticity. A Kruskal-Wallis test showed a modulation of the expression in the visual cortex of m2, m3, m4, m5, α7, ß4, NMDA and GAD65, but only ß4 within the basal forebrain and none of these mRNA within the somatosensory cortex. The two modes of cholinergic enhancement induced different effects on mRNA expression, related to the number of visual conditioning sessions and receptor specificity. This study suggests that the combination of cholinergic enhancement and visual conditioning is specific to the visual cortex and varies between phasic or tonic manipulation of acetylcholine levels.

Comparison of neurotoxic potency between a novel chinbotulinumtoxinA with onabotulinumtoxinA, incobotulinumtoxinA and lanbotulinumtoxinA in rats.

Four botulinumtoxin type A (BoNT/A) products, onabotulinumtoxinA (A/Ona), incobotulinumtoxinA (A/Inco), lanbotulinumtoxinA (A/Lan) and chinbotulinumtoxinA (A/Chin), are applied in the present study, among which A/Chin is newly produced. We aimed to compare the neurotoxic potency of these toxins by the gauge of muscle strength reduction. Furthermore, potential molecular and cellular mechanisms were also explored. According to our data, muscle strengths in the four toxin groups were all significantly decreased after injection for 1 week. A/Chin achieved the most obvious reduction in muscle strength as compared to the other three products at the dose of 0.5 U. However, there was no difference between the four toxins when increased to 2 U. As the toxins wore off, muscle strength recovered to basal level 12 weeks postinjection. We further measured the expression levels of key factors involved in neuromuscular junction stabilization and muscle genesis. Our results showed that nicotinic acetylcholine receptor, myogenic regulatory factors and muscle-specific receptor tyrosine kinase were all significantly upregulated upon BoNT/A treatment. Consistent with the result of muscle strength, A/Chin had the most obvious induction of gene expression. Moreover, we also found local inflammation response following BoNT/A injection. Owing to lack of complexing proteins, both A/Inco and A/Chin stimulated relatively lighter inflammation compared to that of A/Ona and A/Lan groups. In conclusion, our study provided evidence for the efficacy of the novel A/Chin and its similar functional mode to that of A/Ona, A/Inco and A/Lan. In addition, A/Chin has superiority in inducing muscle paralysis and inflammation stimulation, which may indicate faster onset and longer duration of this novel A/Chin.

Myostatin propeptide gene delivery by gene gun ameliorates muscle atrophy in a rat model of botulinum toxin-induced nerve denervation.

AIM: Muscle atrophy is a common symptom after nerve denervation. Myostatin propeptide, a precursor of myostatin, has been documented to improve muscle growth. However, the mechanism underlying the muscle atrophy attenuation effects of myostatin propeptide in muscles and the changes in gene expression are not well established. We investigated the possible underlying mechanisms associated with myostatin propeptide gene delivery by gene gun in a rat denervation muscle atrophy model, and evaluated gene expression patterns. MAIN METHODS: In a rat botulinum toxin-induced nerve denervation muscle atrophy model, we evaluated the effects of wild-type (MSPP) and mutant-type (MSPPD75A) of myostatin propeptide gene delivery, and observed changes in gene activation associated with the neuromuscular junction, muscle and nerve. KEY FINDINGS: Muscle mass and muscle fiber size was moderately increased in myostatin propeptide treated muscles (p<0.05). And enhancement of the gene expression of the muscle regulatory factors, neurite outgrowth factors (IGF-1, GAP43) and acetylcholine receptors was observed. Our results demonstrate that myostatin propeptide gene delivery, especially the mutant-type of MSPPD75A, attenuates muscle atrophy through myogenic regulatory factors and acetylcholine receptor regulation. SIGNIFICANCE: Our data concluded that myostatin propeptide gene therapy may be a promising treatment for nerve denervation induced muscle atrophy.

Clinical application of clustered-AChR for the detection of SNMG.

Myasthenia gravis (MG) is an autoantibody-mediated disease of the neuromuscular junction (NMJ). However, accumulating evidence has indicated that MG patients whose serum anti-acetylcholine receptor (AChR) antibodies are not detectable (serumnegative MG; SNMG) in routine assays share similar clinical features with anti-AChR antibody-positive MG patients. We hypothesized that SNMG patients would have low-affinity antibodies to AChRs that would not be detectable using traditional methods but that might be detected by binding to AChR on the cell membrane, particularly if they were clustered at the high density observed at the NMJ. We expressed AChR subunits with the clustering protein rapsyn (an AChR-associated protein at the synapse) in human embryonic kidney (HEK) cells, and we tested the binding of the antibodies using immunofluorescence. With this approach, AChR antibodies to rapsyn-clustered AChR could be detected in the sera from 45.83% (11/24) of SNMG patients, as confirmed with fluorescence-activated cell sorting (FACS). This was the first application in China of cell-based AChR antibody detection. More importantly, this sensitive (and specific) approach could significantly increase the diagnosis rate of SNMG.

Improved neurological outcome by intramuscular injection of human amniotic fluid derived stem cells in a muscle denervation model.

PURPOSE: The skeletal muscle develops various degrees of atrophy and metabolic dysfunction following nerve injury. Neurotrophic factors are essential for muscle regeneration. Human amniotic fluid derived stem cells (AFS) have the potential to secrete various neurotrophic factors necessary for nerve regeneration. In the present study, we assess the outcome of neurological function by intramuscular injection of AFS in a muscle denervation and nerve anastomosis model. MATERIALS AND METHODS: Seventy two Sprague-Dawley rats weighing 200-250 gm were enrolled in this study. Muscle denervation model was conducted by transverse resection of a sciatic nerve with the proximal end sutured into the gluteal muscle. The nerve anastomosis model was performed by transverse resection of the sciatic nerve followed by four stitches reconnection. These animals were allocated to three groups: control, electrical muscle stimulation, and AFS groups. RESULTS: NT-3 (Neurotrophin 3), BDNF (Brain derived neurotrophic factor), CNTF (Ciliary neurotrophic factor), and GDNF (Glia cell line derived neurotrophic factor) were highly expressed in AFS cells and supernatant of culture medium. Intra-muscular injection of AFS exerted significant expression of several neurotrophic factors over the distal end of nerve and denervated muscle. AFS caused high expression of Bcl-2 in denervated muscle with a reciprocal decrease of Bad and Bax. AFS preserved the muscle morphology with high expression of desmin and acetylcholine receptors. Up to two months, AFS produced significant improvement in electrophysiological study and neurological functions such as SFI (sciatic nerve function index) and Catwalk gait analysis. There was also significant preservation of the number of anterior horn cells and increased nerve myelination as well as muscle morphology. CONCLUSION: Intramuscular injection of AFS can protect muscle apoptosis and likely does so through the secretion of various neurotrophic factors. This protection furthermore improves the nerve regeneration in a long term nerve anastomosis model.

Thiamine deficiency induced neurochemical, neuroanatomical, and neuropsychological alterations: a reappraisal.

Nutritional deficiency can cause, mainly in chronic alcoholic subjects, the Wernicke encephalopathy and its chronic neurological sequela, the Wernicke-Korsakoff syndrome (WKS). Long-term chronic ethanol abuse results in hippocampal and cortical cell loss. Thiamine deficiency also alters principally hippocampal- and frontal cortical-dependent neurochemistry; moreover in WKS patients, important pathological damage to the diencephalon can occur. In fact, the amnesic syndrome typical for WKS is mainly due to the damage in the diencephalic-hippocampal circuitry, including thalamic nuclei and mammillary bodies. The loss of cholinergic cells in the basal forebrain region results in decreased cholinergic input to the hippocampus and the cortex and reduced choline acetyltransferase and acetylcholinesterase activities and function, as well as in acetylcholine receptor downregulation within these brain regions. In this narrative review, we will focus on the neurochemical, neuroanatomical, and neuropsychological studies shedding light on the effects of thiamine deficiency in experimental models and in humans.

Expression of acetylcholine and its receptor in human sympathetic ganglia in primary hyperhidrosis.

BACKGROUND: The pathophysiologic characteristics of primary hyperhidrosis are not well understood and seem to be related to a sympathetic nervous system dysfunction. The resection of thoracic sympathetic chain ganglia is the most effective treatment for hyperhidrosis; however sympathetic ganglia function in normal individuals and in patients with hyperhidrosis is unknown. METHODS: A cross-sectional study, in which 2 groups of 20 subjects were analyzed: the hyperhidrosis group (HYP), comprised of patients with hyperhidrosis who were eligible for thoracic sympathectomy, and the control group (CON) comprised of brain-dead organ donors without a history of hyperhidrosis. For each subject, the following were performed: resection of the third left sympathetic ganglion, measurement of the ganglion's diameter, and immunohistochemical evaluation by quantification of strong and weak expression areas of primary antibodies against acetylcholine and alpha-7 neuronal nicotinic receptor subunit. RESULTS: The presence of a strong alpha-7 subunit expression area was 4.85% in patients with primary hyperhidrosis and 2.34% in controls (p < 0.001), whereas the presence of a weak expression area was 11.48% in the HYP group and 4.59% in the CON group (p < 0.001). Strong acetylcholine expression was found in 4.95% of the total area in the HYP group and in 1.19% in the CON group (p < 0.001), whereas weak expression was found in 18.55% and 6.77% of the HYP and CON groups, respectively (p < 0.001). Furthermore, diameter of the ganglia was 0.71 cm in the HYP group and 0.53 cm in the CON group (p < 0.001). CONCLUSIONS: There is a higher expression of acetylcholine and alpha-7 neuronal nicotinic receptor subunit in the sympathetic ganglia of patients with hyperhidrosis. Furthermore, the diameter of the thoracic sympathetic chain ganglia is larger in such patients.

Cholinergic and GABAergic receptor functional deficit in the hippocampus of insulin-induced hypoglycemic and streptozotocin-induced diabetic rats.

Neurotransmitter receptor functional regulation plays an important role in controlling the excitability and responsiveness of hippocampal neurons. Deregulation of its function is associated with seizure generation, motor deficits, and memory impairment. In the present study we investigated the changes in hippocampal cholinergic and GABA receptor binding and gene expression in insulin-induced hypoglycemic and streptozotocin-induced diabetic rats. Expression of cholinergic enzymes; acetylcholine esterase (AChE) and choline acetyltransferase (ChAT) upregulated and downregulated, respectively, in diabetic group, which was further exacerbated by hypoglycemia. Total muscarinic receptor, muscarinic M1, and GABA maximal binding (B(max)) significantly decreased in hypoglycemic and diabetic rats. In hypoglycemic group, the B(max) showed further decline compared with diabetes. Muscarinic M3 receptor B(max) and gene expression upregulated in hypoglycemic and diabetic group. Alpha7 nicotinic acetylcholine receptor (α7 nAChR) expression significantly downregulated in hypoglycemic and diabetic rats. Gene expression of glutamate decarboxylase (GAD), GABAAα1, and GABAB in hypoglycemic and diabetic rats downregulated, with more significant decrease in hypoglycemic group. Present findings show altered cholinergic, muscarinic, nicotinic receptor expression and thereby function. Decreased GABA receptor expression is associated with decline in GABAergic neurotransmission. Thus cholinergic receptor dysfunction and decreased GABAergic neuroprotective inhibitory function in the hippocampus of hypoglycemic and diabetic rats account for the increased vulnerability of hippocampus predisposing to neuronal damage, which is suggested to contribute to cognitive impairment and memory deficit reported in hypoglycemia and diabetes. Also, recurrent hypoglycemia in diabetes exacerbates the hippocampal dysfunction induced by diabetes, which has clinical significance in diabetes therapy.

Transcriptomic effects of depleted uranium on acetylcholine and cholesterol metabolisms in Alzheimer's disease model.

Some heavy metals, or aluminium, could participate in the development of Alzheimer disease (AD). Depleted uranium (DU), another heavy metal, modulates the cholinergic system and the cholesterol metabolism in the brain of rats, but without neurological disorders. The aim of this study was to determine what happens in organisms exposed to DU that will/are developing the AD. This study was thus performed on a transgenic mouse model for human amyloid precursor protein (APP), the Tg2576 strain. The possible effects of DU through drinking water (20 mg/L) over an 8-month period were analyzed on acetylcholine and cholesterol metabolisms at gene level in the cerebral cortex. The mRNA levels of choline acetyl transferase (ChAT) vesicular acetylcholine transporter (VAChT) and ATP-binding cassette transporter A1 (ABC A1) decreased in control Tg2576 mice in comparison with wild-type mice (respectively -89%, -86% and -44%, p < 0.05). Chronic exposure of Tg2576 mice to DU increased mRNA levels of ChAT (+189%, p < 0.05), VAChT (+120%, p < 0.05) and ABC A1 (+52%, p < 0.05) compared to control Tg2576 mice. Overall, these modifications of acetylcholine and cholesterol metabolisms did not lead to increased disturbances that are specific of AD, suggesting that chronic DU exposure did not worsen the pathology in this experimental model.

Ephexin1 is required for structural maturation and neurotransmission at the neuromuscular junction.

The maturation of neuromuscular junctions (NMJs) requires the topological transformation of postsynaptic acetylcholine receptor (AChR)-containing structures from a simple plaque to an elaborate structure composed of pretzel-like branches. This maturation process results in the precise apposition of the presynaptic and postsynaptic specializations. However, little is known about the molecular mechanisms underlying the plaque-to-pretzel transition of AChR clusters. In this study, we identify an essential role for the RhoGEF ephexin1 in the maturation of AChR clusters. Adult ephexin1(-/-) mice exhibit severe muscle weakness and impaired synaptic transmission at the NMJ. Intriguingly, when ephexin1 expression is deficient in vivo, the NMJ fails to mature into the pretzel-like shape, and such abnormalities can be rescued by re-expression of ephexin1. We further demonstrate that ephexin1 regulates the stability of AChR clusters in a RhoA-dependent manner. Taken together, our findings reveal an indispensible role for ephexin1 in regulating the structural maturation and neurotransmission of NMJs.

T-bet deficiency decreases susceptibility to experimental myasthenia gravis.

T-bet, a tissue-specific transcription factor, controls T helper 1 (Th1) cell differentiation and IFN-production. Given the reciprocal relationship between Th1 and other types of helper T cells, we hypothesized that T-bet impacts multiple helper and regulatory T (Treg) cells, thereby influencing the magnitude of autoimmune disease. We tested this hypothesis in an experimental model of autoimmune myasthenia gravis (EAMG) of mice. Myasthenia gravis (MG) and EAMG are T cell-driven, IgG autoantibody-mediated disorders that destroy muscles by attacking the target antigen acetylcholine receptor (AChR) or other antigens of skeletal muscle at neuromuscular junctions. We show that, compared to wild-type mice, AChR-primed T-bet(-/-) mice are less susceptible to EAMG. This phenotype is associated with a reduction of autoreactive Th1 cells and augmentation of Th2 and Th17 cells as well as an upregulation of Foxp3 expression by T-bet(-/-)CD4(+)CD25(+) Treg cells. Thus, in our model, T-bet not only specifies the Th1 lineage but also has a broad influence on autoreactive Th2, Th17 and Treg cells. These coordinated effects reduce the genesis of pathogenic antibodies and protect against B cell-mediated EAMG.

Myasthenia gravis and autoimmune Addison disease in a patient with thymoma.

The association of thymoma with myasthenia gravis has been well documented. However, the relationship between these two syndromes and Addison disease are very rarely encountered in clinical practice. We report on a 32-year-old man who underwent a resection for thymoma 48 months ago. The diagnosis of Addison disease was made followed by a diagnosis of myasthenia gravis on the basis of a high titer of acetylcholine receptor levels. The treatment of oral prednisolone 7.5 mg/day and oral prostigmine 180 mg/day was initiated. His symptoms and physical signs were improved after this treatment. To our knowledge, this is the fourth reported case of thymoma synchronously associated with myasthenia gravis and Addison disease.

Increased expression of acetylcholine receptors in the diaphragm muscle of MDX mice.

The absence of dystrophin in Duchenne muscular dystrophy (DMD) and in the mutant mdx mouse causes muscle degeneration and disruption of the neuromuscular junction. Based on evidence from the denervation-like properties of these muscles, we assessed the ligand-binding constants of nicotinic acetylcholine receptors (nAChRs) and the mRNA expression of individual subunits in membrane preparations of diaphragm muscles from adult (4-month-old) and aged (20-month-old) control and mdx mice. The concentration of nAChRs as determined by the maximal specific [(125)I]-alpha-bungarotoxin binding (Bmax) in the muscle membranes did not change with aging in both animal strains. When compared to age-matched control groups, the Bmax in mdx muscles was increased by 65% in adults, and by 103% in aged mice with no alteration of toxin affinity for nAChRs. Reverse-transcription polymerase chain reaction assays showed that mRNA transcripts for the nAChR alpha1, gamma, alpha7, and beta2, but not the epsilon subunits, were more abundant in mdx than in control muscles. The results indicate increased expression of extrajunctional nAChRs in the mdx diaphragm and reflect impairment of nAChR regulation in dystrophin-deficient muscles. These observations may be related to the resistance to nondepolarizing muscle relaxants and the high sensitivity to depolarizing agents reported in DMD patients.

Eight genes are required for functional reconstitution of the Caenorhabditis elegans levamisole-sensitive acetylcholine receptor.

Levamisole-sensitive acetylcholine receptors (L-AChRs) are ligand-gated ion channels that mediate excitatory neurotransmission at the neuromuscular junctions of nematodes. They constitute a major drug target for anthelminthic treatments because they can be activated by nematode-specific cholinergic agonists such as levamisole. Genetic screens conducted in Caenorhabditis elegans for resistance to levamisole toxicity identified genes that are indispensable for the biosynthesis of L-AChRs. These include 5 genes encoding distinct AChR subunits and 3 genes coding for ancillary proteins involved in assembly and trafficking of the receptors. Despite extensive analysis of L-AChRs in vivo, pharmacological and biophysical characterization of these receptors has been greatly hampered by the absence of a heterologous expression system. Using Xenopus laevis oocytes, we were able to reconstitute functional L-AChRs by coexpressing the 5 distinct receptor subunits and the 3 ancillary proteins. Strikingly, this system recapitulates the genetic requirements for receptor expression in vivo because omission of any of these 8 genes dramatically impairs L-AChR expression. We demonstrate that 3 alpha- and 2 non-alpha-subunits assemble into the same receptor. Pharmacological analysis reveals that the prototypical cholinergic agonist nicotine is unable to activate L-AChRs but rather acts as a potent allosteric inhibitor. These results emphasize the role of ancillary proteins for efficient expression of recombinant neurotransmitter receptors and open the way for in vitro screening of novel anthelminthic agents.

Na+/K+ ATPase regulates the expression and localization of acetylcholine receptors in a pump activity-independent manner.

Na+/K+ ATPase is a plasma membrane-localized sodium pump that maintains the ion gradients between the extracellular and intracellular environments, which in turn controls the cellular resting membrane potential.Recent evidence suggests that the pump is also localized at synapses and regulates synaptic efficacy.However, its precise function at the synapse is unknown. Here we show that two mutations in the alpha subunit of the eat-6 Na+/K+ ATPase in Caenorhabditis elegans dramatically increase the sensitivity to acetylcholine(Ach) agonists and alter the localization of nicotinic Ach receptors at the neuromuscular junction (NMJ).These defects can be rescued by mutated EAT-6 proteins which lack its pump activity, suggesting the presence of a novel function for Ach signaling. The Na+/K+ ATPase accumulates at postsynaptic sites and appears to surround Ach receptors to maintain rigid clusters at the NMJ. Our findings suggest a pump activity-independent, allele-specific role for Na+/K+ ATPase on postsynaptic organization and synaptic efficacy.

Myotonic dystrophy type 1 coexisting with myasthenia gravis and thymoma.

Myotonic dystrophy type 1 (DM1) is an autosomal-dominant multisystemic disorder that may rarely be associated with benign and malignant neoplasms. Cases of both thymoma and myasthenia gravis in association with DM1 are extremely rare. A literature review revealed only three prior reports. We present a 51-year-old man with a family history of DM1 and fluctuating diplopia and ptosis, who was found to have acetylcholine receptor-binding antibodies, thymoma, and a clinical presentation compatible with ocular myasthenia gravis as well as positive genetic testing for DM1. Needle electromyographic (EMG) study demonstrated diffuse runs of myotonic discharges in multiple muscles, consistent with the diagnosis of DM1. Single-fiber EMG showed both increased jitter and blocking. Due to somatic instability, which has been shown previously in DM1, the myotonin protein kinase (DMPK) gene appears to act as a tumor suppressor. Therefore, abnormal CTG repeat expansions in the gene could lead to the development of thymoma and myasthenia gravis.