A mutation in DOK7 in congenital myasthenic syndrome forms aggresome in cultured cells, and reduces DOK7 expression and MuSK phosphorylation in patient-derived iPS cells.

At the neuromuscular junction, the downstream of tyrosine kinase 7 (DOK7) enhances the phosphorylation of muscle-specific kinase (MuSK) and induces clustering of acetylcholine receptors (AChRs). We identified a patient with congenital myasthenic syndrome (CMS) with two heteroallelic mutations in DOK7, c.653-1G>C in intron 5 and c.190G>A predicting p.G64R in the pleckstrin homology domain. iPS cells established from the patient (CMS-iPSCs) showed that c.653-1G>C caused in-frame skipping of exon 6 (120 bp) and frame-shifting activation of a cryptic splice site deleting seven nucleotides in exon 6. p.G64R reduced the expression of DOK7 to 10% of wild-type DOK7, and markedly compromised AChR clustering in transfected C2C12 myotubes. p.G64R-DOK7 made insoluble aggresomes at the juxtanuclear region in transfected C2C12 myoblasts and COS7 cells, which were co-localized with molecules in the autophagosome system. A protease inhibitor MG132 reduced the soluble fraction of p.G64R-DOK7 and enhanced the aggresome formation of p.G64R-DOK7. To match the differentiation levels between patient-derived and control induced pluripotent stem cells (iPSCs), we corrected c.190G>A (p.G64R) by CRISPR/Cas9 to make isogenic iPSCs while retaining c.653-1G>C (CMS-iPSCsCas9). Myogenically differentiated CMS-iPSCs showed juxtanuclear aggregates of DOK7, reduced expression of endogenous DOK7 and reduced phosphorylation of endogenous MuSK. Another mutation, p.T77M, also made aggresome to a less extent compared with p.G64R in transfected COS7 cells. These results suggest that p.G64R-DOK7 makes aggresomes in cultured cells and is likely to compromise MuSK phosphorylation for AChR clustering.

The clinical and molecular spectrum of ZFYVE26-associated hereditary spastic paraplegia: SPG15.

In the field of hereditary spastic paraplegia (HSP), progress in molecular diagnostics needs to be translated into robust phenotyping studies to understand genetic and phenotypic heterogeneity and to support interventional trials. ZFYVE26-associated hereditary spastic paraplegia (HSP-ZFYVE26, SPG15) is a rare, early-onset complex HSP, characterized by progressive spasticity and a variety of other neurological symptoms. While prior reports, often in populations with high rates of consanguinity, have established a general phenotype, there is a lack of systematic investigations and a limited understanding of age-dependent manifestation of symptoms. Here we delineate the clinical, neuroimaging and molecular features of 44 individuals from 36 families, the largest cohort assembled to date. Median age at last follow-up was 23.8 years covering a wide age range (11-61 years). While symptom onset often occurred in early childhood [median: 24 months, interquartile range (IQR) = 24], a molecular diagnosis was reached at a median age of 18.8 years (IQR = 8), indicating significant diagnostic delay. We demonstrate that most patients present with motor and/or speech delay or learning disabilities. Importantly, these developmental symptoms preceded the onset of motor symptoms by several years. Progressive spasticity in the lower extremities, the hallmark feature of HSP-ZFYVE26, typically presents in adolescence and involves the distal lower limbs before progressing proximally. Spasticity in the upper extremities was seen in 64%. We found a high prevalence of extrapyramidal movement disorders including cerebellar ataxia (64%) and dystonia (11%). Parkinsonism (16%) was present in a subset and showed no sustained response to levodopa. Cognitive decline and neurogenic bladder dysfunction progressed over time in most patients. A systematic analysis of brain MRI features revealed a common diagnostic signature consisting of thinning of the anterior corpus callosum, signal changes of the anterior forceps and non-specific cortical and cerebellar atrophy. The molecular spectrum included 45 distinct variants, distributed across the protein structure without mutational hotspots. Spastic Paraplegia Rating Scale scores, SPATAX Disability Scores and the Four Stage Functional Mobility Score showed moderate strength in representing the proportion of variation between disease duration and motor dysfunction. Plasma neurofilament light chain levels were significantly elevated in all patients (Mann-Whitney U-test, P < 0.0001) and were correlated inversely with age (Spearman's rank correlation coefficient r = -0.65, P = 0.01). In summary, our systematic cross-sectional analysis of HSP-ZFYVE26 patients across a wide age-range, delineates core clinical, neuroimaging and molecular features and identifies markers of disease severity. These results raise awareness to this rare disease, facilitate an early diagnosis and create clinical trial readiness.

Diel rhythm of the inflammatory cytokine il1b in the Japanese medaka (Oryzias latipes) regulated by core components of the circadian clock.

In recent years, studies on circadian control in immunity have been actively conducted in mammals, but little is known about circadian rhythms in the field of fish immunology. In this study, we aimed to analyse the regulation of the diel oscillation of inflammatory cytokine interleukin-1ß (il1b) gene expression by core components of the circadian clock in Japanese medaka (Oryzias latipes). The expression of il1b and clock genes (bmal1 and clock1) in medaka acclimated to a 12:12 light (L): dark (D) cycle showed diel rhythm. Additionally, higher expression of il1b was detected in medaka embryo cells (OLHdrR-e3) overexpressing bmal1 and clock1. A significant decrease in il1b expression was observed in OLHdrR-e3 cells after bmal1 knockdown using morpholino oligos. These changes may be mediated by transcriptional regulation via clock proteins, which target the E-box sequence in the cis-element of il1b as identified using luciferase reporter assays. Moreover, LPS stimulation and pathogenic bacterial infection at different zeitgeber time (ZT) under LD12:12 conditions affected the degree of il1b expression, which showed high and low responsiveness to both immuno-stimulations at ZT2 and ZT14, respectively. These results suggested that fish IL-1ß exhibited diel oscillation regulated by clock proteins, and its responsiveness to immune-stimulation depends on the time of day.

Involvement of brain structures in childhood epilepsy with centrotemporal spikes.

BACKGROUND: We aimed to investigate electroencephalography (EEG)-functional magnetic resonance imaging (fMRI) findings to elucidate the interictal epileptiform discharge (IED)-related functional alterations in deep brain structures and the neocortex in childhood epilepsy with centrotemporal spikes (CECTS). METHODS: Ten children with CECTS (median age 8.2 years), referred to our hospital within a year of onset, were eligible for inclusion. They underwent EEG-fMRI recording during sleep. Llongitudinal evaluations, including medical examinations, intelligence tests, and questionnaires about developmental disabilities, were performed. The initial evaluation was performed at the same time as the EEG-fMRI, and the second evaluation was performed over 2 years after the initial evaluation. RESULTS: Three children were unable to maintain sleep during the EEG-fMRI recording, and the remaining seven children were eligible for further assessment. All patients showed unilateral-dominant centrotemporal spikes during scans. One patient had only positive hemodynamic responses, while the others had both positive and negative hemodynamic responses. All patients showed IED-related hemodynamic responses in the bilateral neocortex. For deep brain structures, IED-related hemodynamic responses were observed in the cingulate gyrus (n = 4), basal ganglia (n = 3), thalamus (n = 2), and default mode network (n = 1). Seizure frequencies at the second evaluation were infrequent or absent, and the longitudinal results of intelligence tests and questionnaires were within normal ranges. CONCLUSIONS: We demonstrated that IEDs affect broad brain areas, including deep brain structures such as the cingulate gyrus, basal ganglia, and thalamus. Deep brain structures may play an important role in the pathophysiology of CECTS.

Risks of ACTH therapy for West syndrome following BCG vaccination.

OBJECTIVE: Bacille de Calmette et Guérin (BCG) is a live vaccine for tuberculosis that is administered to all infants in Japan. Adrenocorticotropic hormone (ACTH) therapy for West syndrome (WS) causes immunosuppression and may result in BCG infection after BCG vaccination. We evaluated the safety of ACTH therapy initiated shortly after BCG vaccination. METHODS: We analyzed patients with WS who received ACTH therapy between 2005 and 2018. We evaluated the interval between BCG and ACTH therapy, and the rate of BCG infection during and after ACTH therapy, by retrospective chart review. RESULTS: Seventy-nine patients were included in the analysis. Twenty-three patients received ACTH therapy prior to BCG vaccination. For the remaining 56 patients, the median interval between BCG vaccination and the start of ACTH therapy (BCG-ACTH interval) was 91.5 (range 14-280) days. The BCG-ACTH interval was shorter in patients with unknown than in those with known etiologies. It was <8 weeks in 13 patients (10 with unknown and 3 with known etiologies). The minimum BCG-ACTH interval was 14 days. Six patients with epileptic spasms received BCG vaccinations because physicians did not recognize their seizures. None of the patients developed BCG infection. CONCLUSION: No patients who received ACTH therapy after BCG, even at an interval of 8 weeks, developed BCG infection. The timing of ACTH therapy initiation should be based on the risk of BCG-related adverse events and the adverse effects of any delay.

Vesicular Polyamine Transporter Mediates Vesicular Storage and Release of Polyamine from Mast Cells.

Mast cells are secretory cells that play an important role in host defense by discharging various intragranular contents, such as histamine and serotonin, upon stimulation of Fc receptors. The granules also contain spermine and spermidine, which can act as modulators of mast cell function, although the mechanism underlying vesicular storage remains unknown. Vesicular polyamine transporter (VPAT), the fourth member of the SLC18 transporter family, is an active transporter responsible for vesicular storage of spermine and spermidine in neurons. In the present study, we investigated whether VPAT functions in mast cells. RT-PCR and Western blotting indicated VPAT expression in murine bone marrow-derived mast cells (BMMCs). Immunohistochemical analysis indicated that VPAT is colocalized with VAMP3 but not with histamine, serotonin, cathepsin D, VAMP2, or VAMP7. Membrane vesicles from BMMCs accumulated spermidine upon the addition of ATP in a reserpine- and bafilomycin A1-sensitive manner. BMMCs secreted spermine and spermidine upon the addition of either antigen or A23187 in the presence of Ca2+, and the antigen-mediated release, which was shown to be temperature-dependent and sensitive to bafilomycin A1 and tetanus toxin, was significantly suppressed by VPAT gene RNA interference. Under these conditions, expression of vesicular monoamine transporter 2 was unaffected, but antigen-dependent histamine release was significantly suppressed, which was recovered by the addition of 1 mm spermine. These results strongly suggest that VPAT is expressed and is responsible for vesicular storage of spermine and spermidine in novel secretory granules that differ from histamine- and serotonin-containing granules and is involved in vesicular release of these polyamines from mast cells.

Change Over Time in Seasonality and Severity of Children Hospitalized With Respiratory Syncytial Virus Infection in Japan.

BACKGROUND: Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis, and in temperate climates, late fall and winter epidemics of bronchiolitis are usually linked to RSV. In recent years in Japan, the RSV infection epidemic has occurred earlier, even during the hot season. This study aimed to evaluate the seasonal variation of RSV-associated hospitalizations over the past decade and the relationship between season and disease severity. METHODS: This was a retrospective single-center study. Hospitalized children were studied between 2011 and 2019. RSV was detected using rapid antigen detection tests. Clinical information was obtained from medical records, and patients were classified by 4 seasons of admission and analyzed for changes over time. RESULTS: Among 3750 children, 945 (25.2%) were RSV-positive. The seasonal proportion of hospitalized children who are RSV-positive showed a peak shift towards summer, with a turning point in 2016. Comparing 2011-2012 and 2018-2019, incidence increased from 6.8% to 46.3% during summer, whereas during fall decreased from 50.0% to 20.7% and decreased from 28.4% to 20.7% during winter ( P < 0.05). A similar trend was observed in the number of children requiring oxygenation in the earlier period; however, after the transition, there was no significant difference between seasons. CONCLUSIONS: Our findings showed that the RSV epidemic shifted to peak during summer until 2016. There was an association between seasonality and severity, such that many younger children were hospitalized during the autumn and winter and required more oxygen; however, after 2016, this difference was no longer observed.

Lenticular nuclei to thalamic ratio on PET is useful for diagnosis of GLUT1 deficiency syndrome.

PURPOSE: To establish an objective method of [18F]-fluorodeoxyglucose positron emission tomography (FDG-PET) that can assist in the diagnosis of glucose transporter 1 deficiency syndrome (GLUT1-DS). METHODS: FDG-PET was performed in 8 patients with a mean age of 12.5 years (range, 2-22 years) with GLUT1-DS. Their PET findings were compared with those of 45 controls with a mean age of 11.2 years (range, 2-21 years) by statistical parametric mapping (SPM12, Welcome Neurological Institute). The controls had epilepsy of unknown etiology and normal MRI findings. The age-adjusted ratios of mean radioactivities in regions of interest (ROIs) of bilateral lenticular nuclei, thalami, and the whole cerebral cortex were also measured. The sensitivities and specificities of the ratios for the differential diagnosis of GLUT1-DS were also determined. RESULTS: SPM showed significantly decreased uptake in bilateral thalami and increased uptake in bilateral lenticular nuclei in patients with GLUT1-DS. There were no areas in the cerebral cortex with significant differences between patients and controls. On ROI analysis, by setting the cut-off value of the age-adjusted lenticular nuclei/thalami radioactivity ratio to 1.54, patients with GLUT1-DS were differentiated from controls with sensitivity of 1.00 and specificity of 0.98. CONCLUSION: The age-adjusted lenticular nuclei/thalami radioactivity ratio on PET can distinguish patients with GLUT1-DS from patients with epilepsy of unknown etiology with high sensitivity and specificity. It is important to pay attention to the metabolism of the lenticular nuclei and thalami on PET for the diagnosis of GLUT1-DS.

Lacosamide for children with paroxysmal kinesigenic dyskinesia.

OBJECTIVES: This study was performed to evaluate the efficacy and tolerability of lacosamide (LCM) for paroxysmal kinesigenic dyskinesia (PKD) in children. METHODS: We retrospectively reviewed the medical charts of pediatric PKD patients (aged <16 years) treated with LCM. Data regarding demographic characteristics, proline-rich transmembrane protein 2 (PRRT2) gene variant, clinical features of PKD, dose of LCM, efficacy, and adverse events were recorded. RESULTS: Four eligible patients (3 males, 1 female) were identified, with an age of onset ranging from 8.3 to 14.7 years. PRRT2 variant was evaluated in three children and a c.649dupC variant was identified in one child with a positive family history. Attacks were bilateral in three children and left-sided in one. Two children had a family history of PKD and one child had a family history of benign infantile epilepsy. Treatment with carbamazepine failed in two children due to drowsiness and auditory disturbance. The initial dose of LCM was 50 mg/day in three children and 100 mg/day in one. All patients were attack-free within a few days. The maintenance dose was mostly similar to the initial dose. No adverse events related to LCM were reported during follow-up. CONCLUSIONS: LCM is an effective and well-tolerated treatment for PKD in children, and low-dose treatment may be viable.

Spatially Arranged Sparse Recurrent Neural Networks for Energy Efficient Associative Memory.

The development of hardware neural networks, including neuromorphic hardware, has been accelerated over the past few years. However, it is challenging to operate very large-scale neural networks with low-power hardware devices, partly due to signal transmissions through a massive number of interconnections. Our aim is to deal with the issue of communication cost from an algorithmic viewpoint and study learning algorithms for energy-efficient information processing. Here, we consider two approaches to finding spatially arranged sparse recurrent neural networks with the high cost-performance ratio for associative memory. In the first approach following classical methods, we focus on sparse modular network structures inspired by biological brain networks and examine their storage capacity under an iterative learning rule. We show that incorporating long-range intermodule connections into purely modular networks can enhance the cost-performance ratio. In the second approach, we formulate for the first time an optimization problem where the network sparsity is maximized under the constraints imposed by a pattern embedding condition. We show that there is a tradeoff between the interconnection cost and the computational performance in the optimized networks. We demonstrate that the optimized networks can achieve a better cost-performance ratio compared with those considered in the first approach. We show the effectiveness of the optimization approach mainly using binary patterns and apply it also to gray-scale image restoration. Our results suggest that the presented approaches are useful in seeking more sparse and less costly connectivity of neural networks for the enhancement of energy efficiency in hardware neural networks.

Role of peptide transporter 2 and MAPK signaling pathways in the innate immune response induced by bacterial peptides in alveolar epithelial cells.

AIMS: The innate immune response induced by bacterial peptidoglycan peptides, such as γ-d-glutamyl-meso-diaminopimelic acid (iE-DAP), is an important host defense system. However, little is known about the innate immune response in the lung alveolar region. In this study, we examined induction of the innate immune response by iE-DAP in human alveolar epithelial cell lines, NCI-H441 (H441) and A549. MAIN METHODS: Induction of the innate immune response was evaluated by measuring the mRNA expression of cytokines and their release into the culture medium. KEY FINDINGS: iE-DAP treatment increased the mRNA expression of interleukin (IL)-6 and IL-8, and increased release of these pro-inflammatory cytokines into the culture medium in H441 cells, but not in A549 cells. Lack of release of these cytokines in A549 cells may have been due to lack of peptide transporter 2 (PEPT2) function. Intracellular nucleotide-binding oligomerization domain 1 (NOD1) recognizes iE-DAP and activates downstream signaling pathways to initiate the immune response. Therefore, the role of mitogen-activated protein kinase (MAPK) signaling pathways was examined in H441 cells. As a result of inhibition studies, receptor-interacting serine/threonine-protein kinase 2 and MAPK signaling pathways, such as p38 MAPK and extracellular signal-regulated kinase, but not c-Jun N-terminal kinase, were determined to be involved in the innate immune response in H441 cells. In addition, the nuclear factor κB pathway also played a role in the innate immune response. SIGNIFICANCE: These findings indicated that the innate immune response induced by bacterial peptides could occur in a PEPT2- and NOD1-dependent manner in alveolar epithelial cells.

Paroxysmal nonepileptic events in children with epilepsy.

OBJECTIVE: The aim of this study was to clarify the characteristics of paroxysmal nonepileptic events (PNEs) suspected as being epileptic seizures by families of children with epilepsy. METHODS: The video-EEG (vEEG) recordings of habitual paroxysmal events in children with epilepsy at Nagoya University Hospital between October 2006 and January 2016 were reviewed. Based on the doctor's suspicion before the vEEG, the PNEs were divided into two groups that included PNEs suspected as epileptic seizures and PNEs suspected as PNEs. PNEs in the former group were classified based on the suspected seizure type. RESULTS: Of 886 habitual paroxysmal events, vEEG confirmed that 83 events (68 children) were PNEs. The median age of the 68 children was 3.2 years. Concurrent epilepsies included focal epilepsies (n=33), infantile spasms (n=16), and other types (n=19). The most common types of PNEs were sleep myoclonus (n=11), followed by stereotypies (n=9), awake myoclonus (n=8), paroxysmal ocular deviations (PODs, n=8), and tonic posturing (n=8). Even after direct observation or video viewing, the doctors suspected epileptic seizures in all three of the PODs and two of the tonic posturing children. Before the vEEG, however, the accurate visual information led to the speculation that the four psychogenic and two sleep myoclonus events were all PNEs. Myoclonus, stereotypies, and head drops were often misdiagnosed as epileptic spasms, while PODs and tonic posturing were often misdiagnosed as focal seizures with motor components. Additionally, staring and motion arrest during a drowsy state were often misdiagnosed as focal dyscognitive seizures. Seven of eight patients with PODs had epileptic spasms that were concurrent with epileptic seizures. A diffuse cerebral lesion or reduced visual acuity was seen in seven patients with PODs. CONCLUSION: We re-emphasize that vEEG is essential for accurate diagnosis and provides evidence for listing POD in the differential diagnosis of oculomotor paroxysmal events.

The effects of co-medications on lamotrigine clearance in Japanese children with epilepsy.

PURPOSE: Although it has been reported that some antiepileptic drugs have inducing or inhibiting effects on lamotrigine (LTG) clearance, whether they have the same effects in Asian epilepsy patients as in those in other countries has not been clarified, especially in children. The aim of this study was to determine the effects of co-medications on LTG clearance in Japanese children with epilepsy. METHODS: A total of 342 routine serum concentration measurements of LTG in 102 Japanese epilepsy patients under 20years of age were reviewed. The dose-corrected concentration (DCC) of LTG was calculated as [concentration]/[dose/(body weight)], and the DCC of LTG was compared by co-medication. The difference in the DCC of LTG was compared between patients with and without valproic acid (VPA) and between those with and without drugs inducing glucuronic acid conjugation (phenytoin (PHT), carbamazepine (CBZ), and phenobarbital (PB)). RESULTS: The DCC of LTG was significantly higher in patients on VPA and significantly lower in patients on drugs inducing glucuronic acid conjugation than in patients on LTG monotherapy. The DCC of LTG was significantly higher in patients on CBZ than in patients on PHT or PB. There was no correlation between the DCC of LTG and the concentration of VPA or metabolic inducers within the therapeutic range. Other antiepileptic drugs including clobazam, clonazepam, zonisamide, and levetiracetam had little effect on LTG concentration. CONCLUSION: LTG concentration changes dramatically with concomitant antiepileptic drugs in Japanese children, as previously reported from other countries, and special attention is required. Although the dose of LTG should be adjusted when starting or discontinuing VPA or metabolic inducers, no adjustment is needed when changing the dose of VPA or metabolic inducers in the therapeutic range.

Parsimonious description for predicting high-dimensional dynamics.

When we observe a system, we often cannot observe all its variables and may have some of its limited measurements. Under such a circumstance, delay coordinates, vectors made of successive measurements, are useful to reconstruct the states of the whole system. Although the method of delay coordinates is theoretically supported for high-dimensional dynamical systems, practically there is a limitation because the calculation for higher-dimensional delay coordinates becomes more expensive. Here, we propose a parsimonious description of virtually infinite-dimensional delay coordinates by evaluating their distances with exponentially decaying weights. This description enables us to predict the future values of the measurements faster because we can reuse the calculated distances, and more accurately because the description naturally reduces the bias of the classical delay coordinates toward the stable directions. We demonstrate the proposed method with toy models of the atmosphere and real datasets related to renewable energy.

Identification of a mammalian vesicular polyamine transporter.

Spermine and spermidine act as neuromodulators upon binding to the extracellular site(s) of various ionotropic receptors, such as N-methyl-d-aspartate receptors. To gain access to the receptors, polyamines synthesized in neurons and astrocytes are stored in secretory vesicles and released upon depolarization. Although vesicular storage is mediated in an ATP-dependent, reserpine-sensitive fashion, the transporter responsible for this process remains unknown. SLC18B1 is the fourth member of the SLC18 transporter family, which includes vesicular monoamine transporters and vesicular acetylcholine transporter. Proteoliposomes containing purified human SLC18B1 protein actively transport spermine and spermidine by exchange of H(+). SLC18B1 protein is predominantly expressed in the hippocampus and is associated with vesicles in astrocytes. SLC18B1 gene knockdown decreased both SLC18B1 protein and spermine/spermidine contents in astrocytes. These results indicated that SLC18B1 encodes a vesicular polyamine transporter (VPAT).

Thalamic lesions in acute encephalopathy with biphasic seizures and late reduced diffusion.

BACKGROUND: We aimed to assess the characteristics of thalamic lesions in children with acute encephalopathy with biphasic seizures and late reduced diffusion. METHODS: Using the Tokai Pediatric Neurology Society database, we identified and enrolled 18 children with acute encephalopathy with biphasic seizures and late reduced diffusion from 2008 to 2010. Using diffusion-weighted images, we identified patients with thalamic lesions and compared their clinical factors with those of patients without thalamic lesions. We analyzed the time sequence of thalamic, sucortical, and cortical lesions. To study the topography of thalamic lesions, we divided the thalamus into five sections: anterior, medial, anterolateral, posterolateral, and posterior. Subsequently, we analyzed the relationship between the topography of thalamic lesions and the presence of central-sparing. RESULTS: Seven children presented with symmetrical thalamic lesions associated with bilateral subcortical or cortical lesions. No statistical difference in the clinical features was observed between individuals with and without thalamic lesions. These lesions were observed only when subcortical or cortical lesions were present. In 5 children, thalamic lesions were present in bilateral anterior or anterolateral sections and were associated with subcortical or cortical lesions in bilateral frontal lobes with central-sparing. In the other two children, thalamic lesions were extensive and accompanied by diffuse subcortical and cortical lesions without central-sparing. CONCLUSION: Thalamic lesions in patients with acute encephalopathy with biphasic seizures and late reduced diffusion involve the anterior sections. The thalamocortical network may play a role in development of thalamic lesions in patients with acute encephalopathy with biphasic seizures and late reduced diffusion.