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Myasthenia gravis (MG) is an autoimmune neuromuscular disease characterized by fatigable skeletal muscle weakness with a fluctuating unpredictable course. One main concern in MG is the lack of objective biomarkers to guide individualized treatment decisions. Specific circulating serum microRNAs (miRNAs) miR-30e-5p, miR-150-5p and miR-21-5p levels have been shown to correlate with clinical course in specific MG patient subgroups. The aim of our study was to better characterize these miRNAs, regardless of the MG subgroup, at an early stage from diagnosis and determine their sensitivity and specificity for MG diagnosis, as well as their predictive power for disease relapse. Serum levels of these miRNAs in 27 newly diagnosed MG patients were compared with 245 healthy individuals and 20 patients with non-MG neuroimmune diseases. Levels of miR-30e-5p and miR-150-5p significantly differed between MG patients and healthy controls; however, no difference was seen compared with patients affected by other neuroimmune diseases. High levels of miR-30e-5p predicted MG relapse (p = 0.049) with a hazard ratio of 2.81. In summary, miR-150-5p is highly sensitive but has low specificity for MG, while miR-30e-5p has the greatest potential as a predictive biomarker for the disease course in MG, regardless of subgroup.

1. Skin nerve sympathetic activity was recorded simultaneously from the following pairs of nerves: left and right median, median and peroneal, left and right peroneal, posterior cutaneous antebrachial and superficial radial, posterior cutaneous antebrachial and median. The recordings were performed on healthy subjects exposed to different ambient temperatures. Electrodermal responses and pulse plethysmograms were recorded from the neural innervation zones. 2. Vasoconstriction impulse bursts recorded simultaneously from the median and peroneal nerves during exposure to a cold environment showed a striking similarity with respect to the timing and strength of individual bursts. A similar strong correlation was observed also among sudomotor bursts recorded simultaneously from the posterior cutaneous antebrachial and superficial radial nerve during exposure to a warm environment. 3. On some occasions, such as during exposure to a moderately warm environment or emotional stress, a temporal correlation was also observed between vasoconstrictor bursts recorded from the median and sudomotor bursts recorded simultaneously from the posterior cutaneous antebrachial nerve. 4. The double nerve recordings provided evidence that in the distal glabrous skin areas reflex thermoregulatory functions are mainly executed via vasoconstrictor fibres whereas sudomotor fibres are brought into action only at relatively high temperature. On the contrary, in the hairy skin on the dorsal side of forearm and hand reflex thermoregulation is to a large extent executed via sudomotor fibres.

1. Recordings of multiunit sympathetic activity were made from human nerve fascicles supplying hairy and glabrous skin of the extremities in healthy subjects exposed to different ambient temperatures. Sudomotor and vasomotor events accompanying the neural activity were monitored by simultaneous recordings of electrodermal and pulse plethysmographic events (Pleth) in the neural innervation zones. 2. By exposing the subject to warm (43 degrees C) or cold (15 degrees C) environments, it was possible to obtain a selective activation of either the sudomotor or the vasoconstrictor neural system, respectively, with suppression of spontaneous activity in the other system. 3. Bursts of both vasoconstrictor and sudomotor nerve activity were found to occur at certain preferred intervals which were integer multiples of a period of about 0 . 6 sec (100 cycles/min). With high sudomotor or vasoconstrictor tone the 100 cycles/min rhythm was prominent but with decreasing tone slower subharmonic rhythms prevailed. Respiratory rhythms were also discerned as well as slower rhythms attributable to oscillatory tendencies in thermoregulatory servos. 4. Vasoconstrictor bursts had longer mean duration than sudomotor bursts, a finding attributed to a slower conduction velocity of vasoconstrictor as compared to sudomotor impulses. 5. With increasing incidence of bursts transient electrodermal or plethysmographic responses following individual bursts merged, and thus the fast neural rhythms were not discernible in either the electrodermal or Pleth traces. Given increments in firing rate of nerves produced less additional vasoconstriction at high than at low firing rates. The rhythm generating mechanisms may help to restrict rates of individual fibres to the low range which provides high gain in the neuroeffector transfer functions.

The neuromuscular junction (NMJ) serves as a model for understanding the mechanisms that determine communication between neurons and their target cells. Disorders of the NMJ can be either autoimmune or genetic (hereditary). The autoimmune disorder myasthenia gravis (MG) is caused by antibodies against the presynaptic nerve terminal or the postsynaptic muscle membrane, which make up the NMJ. The most common antibodies are directed against the acetylcholine receptor (AChR) or muscle specific tyrosine kinase (MuSK). An alternative to expand on preclinical in-vivo methods for studying mechanisms underlying diseases of neuromuscular transmission is to apply physiologic in-vitro models that would allow tissue-tissue as well as cell-cell interactions. A system that would allow cell-cell interactions in a biological fashion is the micro-electrode array (MEA) chip that allows co-culturing of motor neurons and muscle cells.

The primary hypothesis is that the suggested MEA can be used in creating a reliable model for healthy and diseased NMJ, allowing for manipulations and treatment assays. The secondary hypothesis is that small non-coding RNA, so called microRNAs (miRNA) have a specific role in neuromuscular transmission and in MG.

Study I demonstrated a method of long-term muscle cell culture on the MEA chips, which allows us to trace the development of muscle cells through the observation of their electrical activity at subcellular resolution. The maturation of skeletal muscle tissue was accompanied by a gradual increase in the amplitude and frequency of extracellular individual electrical spikes. The mature muscle tissue demonstrated the steady electrical activity with synchronized spike propagation in different directions across the chip.

Study II showed a specific upregulated profile of miRNAs in the muscles of MuSK antibody seropositive MG mice. Transfection of these miRNAs, miR-1933 and miR-1930, promoted downregulation of several proteins and further confirmation with qPCR revealed a specific blocking of IMPA1 and MRPL27, which are involved in intracellular signal transduction and mitochondrial biogenesis in skeletal muscles.

Study III revealed no correlation between the morphology of skeletal muscle cells and their electrical activity at an early developmental stage. However, the application of recombinant rat agrin increased the number of AChRs clusters in the culture of skeletal muscle and promoted a higher degree of spontaneous activity.

MuSK antibody seropositive (MuSK+) Myasthenia Gravis (MG) typically affects skeletal muscles of the bulbar area, including the omohyoid muscle, causing focal fatigue, weakness and atrophy. The profile of circulating extracellular microRNA (miRNA) is changed in MuSK + MG, but the intracellular miRNA profile in skeletal muscles of MuSK + MG and MuSK + experimental autoimmune MG (EAMG) remains unknown. This study elucidated the intracellular miRNA profile in the omohyoid muscle of mice with MuSK + EAMG. The levels of eleven mouse miRNAs were elevated and two mouse miRNAs were reduced in muscles of MuSK + EAMG mice. Transient expression of miR-1933-3p and miR-1930-5p in mouse muscle (C2C12) cells revealed several downregulated genes, out of which five had predicted binding sites for miR-1933-3p. The mRNA expression of mitochondrial ribosomal protein L27 (Mrpl27) and Inositol monophosphatase I (Impa1) was reduced in miR-1933-3p transfected C2C12 cells compared to control cells (p = 0.032 versus p = 0.020). Further, transient expression of miR-1933-3p reduced Impa1 protein accumulation in C2C12 cells. These findings provide novel insights of dysregulated miRNAs and their intracellular pathways in muscle tissue afflicted with MuSK + EAMG, providing a possible link to mitochondrial dysfunction and muscle atrophy observed in MuSK + MG.

Introduction/Aims: The motor unit size index (MUSIX) may provide insight into reinnervation patterns in diseases such as amyotrophic lateral sclerosis (ALS). However, it is not known whether MUSIX detects clinically relevant changes in reinnervation, or if all muscles manifest changes in MUSIX in response to reinnervation after motor unit loss.

Methods: Fifty-seven patients with ALS were assessed at 3-month intervals for 12 months in four centers. Muscles examined were abductor pollicis brevis, abductor digiti minimi, biceps brachii, and tibialis anterior. Results were split into two groups: muscles with increases in MUSIX and those without increases. Longitudinal changes in MUSIX, motor unit number index (MUNIX), compound muscle action potential amplitude, and Medical Research Council strength score were investigated.

Results: One hundred thirty-three muscles were examined. Fifty-nine percent of the muscles exhibited an increase in MUSIX during the study. Muscles with MUSIX increases lost more motor units (58% decline in MUNIX at 12 months, P <.001) than muscles that did not increase MUSIX (34.6% decline in MUNIX at 12 months, P <.001). However, longitudinal changes in muscle strength were similar. When motor unit loss was similar, the absence of a MUSIX increase was associated with a significantly greater loss of muscle strength (P =.002).

Discussion: MUSIX increases are associated with greater motor unit loss but relative preservation of muscle strength. Thus, MUSIX appears to be measuring a clinically relevant response that can provide a quantitative outcome measure of reinnervation in clinical trials. Furthermore, MUSIX suggests that reinnervation may play a major role in determining the progression of weakness.

Modern neuromuscular electrodiagnosis (EDX) and neuromuscular ultrasound (NMUS) require a universal language for effective communication in clinical practice and research and, in particular, for teaching young colleagues. Therefore, the AANEM and the IFCN have decided to publish a joint glossary as they feel the need for an updated terminology to support educational activities in neuromuscular EDX and NMUS in all parts of the world. In addition NMUS has been rapidly progressing over the last years and is now widely used in the diagnosis of disorders of nerve and muscle in conjunction with EDX. This glossary has been developed by experts in the field of neuromuscular EDX and NMUS on behalf of the AANEM and the IFCN and has been agreed upon by electronic communication between January and November 2019. It is based on the glossaries of the AANEM from 2015 and of the IFCN from 1999. The EDX and NMUS terms and the explanatory illustrations have been updated and supplemented where necessary. The result is a comprehensive glossary of terms covering all fields of neuromuscular EDX and NMUS. It serves as a standard reference for clinical practice, education and research worldwide. (C) 2020 the American Association of Neuromuscular & Electrodiagnostic Medicine and the International Federation of Clinical Neurophysiology.

Modern neuromuscular electrodiagnosis (EDX) and neuromuscular ultrasound (NMUS) require a universal language for effective communication in clinical practice and research and, in particular, for teaching young colleagues. Therefore, the AANEM and the IFCN have decided to publish a joint glossary as they feel the need for an updated terminology to support educational activities in neuromuscular EDX and NMUS in all parts of the world. In addition NMUS has been rapidly progressing over the last years and is now widely used in the diagnosis of disorders of nerve and muscle in conjunction with EDX. This glossary has been developed by experts in the field of neuromuscular EDX and NMUS on behalf of the AANEM and the IFCN and has been agreed upon by electronic communication between January and November 2019. It is based on the glossaries of the AANEM from 2015 and of the IFCN from 1999. The EDX and NMUS terms and the explanatory illustrations have been updated and supplemented where necessary. The result is a comprehensive glossary of terms covering all fields of neuromuscular EDX and NMUS. It serves as a standard reference for clinical practice, education and research worldwide.

ALG2 mutations are extremely rare causes of congenital myasthenic syndromes (CMS). The clinical phenotype and treatment response is therefore not well described. We present the case of a baby who immediately after birth presented with pronounced truncal hypotonia, proximal muscle weakness and feeding difficulties. Single fibre electromyography showed neuromuscular transmission failure and salbutamol and ephedrine treatment improved both muscle weakness and neuromuscular transmission. Genetic analysis revealed a likely pathogenic variant c.1040del, p.(Gly347Valfs*27) in exon 2 and a variant of uncertain significance, c.239G>A, p.(Gly80Asp) in exon 1 of the ALG2 gene. Western blot in whole cell lysates of HEK293 cells transfected with p.Gly80Asp, or p.Gly347Valfs*27 expression constructs indicated that p.Gly347Valfs*27 is likely a null allele and p.Gly80Asp is pathogenic through marked reduction of ALG2 expression. This case highlights the utility of functional studies in clarifying variants of unknown significance, in suspected cases of CMS. (C) 2021 The Author(s). Published by Elsevier B.V.

Background

Prolonged seizures generate cerebral hypoxia and increased intracranial pressure, resulting in an increased risk of neurological deterioration, increased long-term morbidity, and shorter survival. Seizures should be recognized early and treated promptly.

The aim of the study was to investigate the occurrence of postoperative seizures in patients undergoing craniotomy for primary brain tumors and to determine if non-convulsive seizures could explain some of the postoperative neurological deterioration that may occur after surgery.

Methods

A single-center prospective study of 100 patients with suspected glioma. Participants were studied with EEG and video recording for at least 24 h after surgery.

Results

Seven patients (7%) displayed seizure activity on EEG recording within 24 h after surgery and another two patients (2%) developed late seizures. One of the patients with early seizures also developed late seizures. In five patients (5%), there were non-convulsive seizures. Four of these patients had a combination of clinically overt and non-convulsive seizures and in one patient, all seizures were non-convulsive. The non-convulsive seizures accounted for the majority of total seizure time in those patients. Non-convulsive seizures could not explain six cases of unexpected postoperative neurological deterioration. Postoperative ischemic lesions were more common in patients with early postoperative seizures.

Conclusions

Early seizures, including non-convulsive, occurred in 7% of our patients. Within this group, non-convulsive seizure activity had longer durations than clinically overt seizures, but only 1% of patients had exclusively non-convulsive seizures. Seizures were not associated with unexpected neurological deterioration.

Neurochondrin (NCDN) is a cytoplasmatic neural protein of importance for neural growth, glutamate receptor (mGluR) signaling, and synaptic plasticity. Conditional loss of Ncdn in mice neural tissue causes depressive-like behaviors, impaired spatial learning, and epileptic seizures. We report on NCDN missense variants in six affected individuals with variable degrees of developmental delay, intellectual disability (ID), and seizures. Three siblings were found homozygous for a NCDN missense variant, whereas another three unrelated individuals carried different de novo missense variants in NCDN. We assayed the missense variants for their capability to rescue impaired neurite formation in human neuroblastoma (SH-SY5Y) cells depleted of NCDN. Overexpression of wild-type NCDN rescued the neurite-phenotype in contrast to expression of NCDN containing the variants of affected individuals. Two missense variants, associated with severe neurodevelopmental features and epilepsy, were unable to restore mGluR5-induced ERK phosphorylation. Electrophysiological analysis of SH-SY5Y cells depleted of NCDN exhibited altered membrane potential and impaired action potentials at repolarization, suggesting NCDN to be required for normal biophysical properties. Using available transcriptome data from human fetal cortex, we show that NCDN is highly expressed in maturing excitatory neurons. In combination, our data provide evidence that bi-allelic and de novo variants in NCDN cause a clinically variable form of neurodevelopmental delay and epilepsy, highlighting a critical role for NCDN in human brain development.

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate important intracellular biological processes. In myasthenia gravis (MG), a disease-specific pattern of elevated circulating miRNAs has been found, and proposed as potential biomarkers. These elevated miRNAs include miR-150-5p, miR-21-5p, and miR-30e-5p in acetylcholine receptor antibody seropositive (AChR+) MG and miR-151a-3p, miR-423-5p, let-7a-5p, and let-7f-5p in muscle-specific tyrosine kinase antibody seropositive (MuSK+) MG. In this study, we examined the regulation of each of these miRNAs using chromatin immunoprecipitation sequencing (ChIP-seq) data from the Encyclopedia of DNA Elements (ENCODE) to gain insight into the transcription factor pathways that drive their expression in MG. Our aim was to look at the transcription factors that regulate miRNAs and then validate some of those in vivo with cell lines that have sufficient expression of these transcription factors This analysis revealed several transcription factor families that regulate MG-specific miRNAs including the Forkhead box or the FOXO proteins (FoxA1, FoxA2, FoxM1, FoxP2), AP-1, interferon regulatory factors (IRF1, IRF3, IRF4), and signal transducer and activator of transcription proteins (Stat1, Stat3, Stat5a). We also found binding sites for nuclear factor of activated T-cells (NFATC1), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappa B), early growth response factor (EGR1), and the estrogen receptor 1 (ESR1). AChR+ MG miRNAs showed a stronger overall regulation by the FOXO transcription factors, and of this group, miR-21-5p, let-7a, and let 7f were found to possess ESR1 binding sites. Using a murine macrophage cell line, we found activation of NF-kappa B -mediated inflammation by LPS induced expression of miR-21-5p, miR-30e-5p, miR-423-5p, let-7a, and let-7f. Pre-treatment of cells with the anti-inflammatory drugs prednisone or deflazacort attenuated induction of inflammation-induced miRNAs. Interestingly, the activation of inflammation induced packaging of the AChR+-specific miRNAs miR-21-5p and miR-30e-5p into exosomes, suggesting a possible mechanism for the elevation of these miRNAs in MG patient serum. In conclusion, our study summarizes the regulatory transcription factors that drive expression of AChR+ and MuSK+ MG-associated miRNAs. Our findings of elevated miR-21-5p and miR-30e-5p expression in immune cells upon inflammatory stimulation and the suppressive effect of corticosteroids strengthens the putative role of these miRNAs in the MG autoimmune response.

OBJECTIVE: The aim was to characterize the electrophysiological features and plasma biomarkers of critical illness polyneuropathy (CIN) and myopathy (CIM) in coronavirus disease 2019 (COVID-19) patients with intensive care unit acquired weakness (ICUAW).

METHODS: An observational ICU cohort study including adult patients admitted to the ICU at Uppsala University Hospital, Uppsala, Sweden, from March 13th to June 8th 2020. We compared the clinical, electrophysiological and plasma biomarker data between COVID-19 patients who developed CIN/CIM and those who did not. Electrophysiological characteristics were also compared between COVID-19 and non-COVID-19 ICU patients.

RESULTS: 111 COVID-19 patients were included, 11 of whom developed CIN/CIM. Patients with CIN/CIM had more severe illness; longer ICU stay, more thromboembolic events and were more frequently treated with invasive ventilation for longer than 2 weeks. In particular CIN was more frequent among COVID-19 patients with ICUAW (50%) compared with a non-COVID-19 cohort (0%, p = 0.008). Neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAp) levels were higher in the CIN/CIM group compared with those that did not develop CIN/CIM (both p = 0.001) and correlated with nerve amplitudes.

CONCLUSIONS: CIN/CIM was more prevalent among COVID-19 ICU patients with severe illness.

SIGNIFICANCE: COVID-19 patients who later developed CIN/CIM had significantly higher NfL and GFAp in the early phase of ICU care, suggesting their potential as predictive biomarkers for CIN/CIM.

Introduction

To reduce myasthenia gravis (MG) patient risk of immunosuppressant (IS) exposure adverse events (AEs), such as infections and malignancies, and to reduce treatment burden, international guidelines recommend decreasing IS dose in stable MG patients.

Methods

Online surveys were conducted of self‐identified MG patients and MG physician experts about the importance of IS dose reduction for MG patients who achieve prolonged periods of disease stability.

Results

Eighty‐four percent of MG patients (n = 283) and 100% of physicians (n = 45) were concerned about long‐term IS‐associated AEs. Although both groups favored attempting IS reduction, they raised concerns including MG relapse, hospitalization, and uncertainty about the future. Presented with an estimated 12% significant relapse rate with IS dose reduction, 76% of patients would be willing to enroll in a randomized IS dose reduction trial.

Discussion

Patients and physicians favor considering IS dose reduction but are also concerned about potential negative sequelae.

Objectives: To estimate the jitter parameters (single-fiber electromyography) in myasthenia gravis patients mostly by electrical activation in Frontalis, Orbicularis Oculi, and Extensor Digitorum muscles using a concentric needle electrode.

Methods: Between 2009 and 2019, a total of 97 myasthenia gravis patients, 52 male, and mean age 54 years were included.

Results: Any abnormal jitter parameter in individual muscles was 90.5% (Frontalis), 88.5% (Orbicularis Oculi), and 86.6% (Extensor Digitorum). Any jitter parameter combining Orbicularis Oculi and Frontalis muscle was abnormal in 100% for the ocular, and in 92.9% for the generalized myasthenia gravis. The most abnormal muscle was Orbicularis Oculi for the generalized, and Frontalis for the ocular myasthenia gravis. The decrement was abnormal in 78.4%, 85.9% for the generalized, and 25% for the ocular myasthenia gravis. The mean jitter ranged from 14.2 to 86 mu s (mean 33.3 mu s) for the ocular myasthenia gravis and from 14.4 to 220.4 mu s (mean 66.3 mu s) for the generalized myasthenia gravis. The antibody titers tested positive in 86.6%, 91.8% for the generalized, and 50% for the ocular myasthenia gravis. Thymectomy was done in 48.5%, thymoma was found in 19.6%, and myasthenic crisis occurred by 21.6%.

Conclusion: The jitter parameters achieved a 100% abnormality in ocular myasthenia gravis if both the Orbicularis Oculi and Frontalis muscles were tested. There was a high jitter abnormality in generalized myasthenia gravis cases with one muscle tested, with about a 2% increase in sensitivity when a second is added. Concentric needle electrode jitter had high sensitivity similar to the single fiber electrode (93.8%), followed by antibody titers (86.6%), and abnormal decrement (78.4%).