村松 一洋

Abstract Static encephalopathy of childhood with neurodegeneration in adulthood/β-propeller protein-associated neurodegeneration is a neurodegenerative disorder with brain iron accumulation caused by the variants of WDR45, a core autophagy-related gene that encodes WIPI4. However, the pathophysiology of the disease, particularly the function of WDR45/WIPI4 in iron metabolism, is largely unknown. As no other variants of core autophagy-related genes show abnormalities in iron metabolism, the relation between autophagy and iron metabolism remains to be elucidated. Since iron deposition in the brain is the hallmark of static encephalopathy of childhood with neurodegeneration in adulthood/β-propeller protein-associated neurodegeneration, iron chelation therapy has been attempted, but it was found to worsen the symptoms; thus, the establishment of a curative treatment is essential. Here, we evaluated autophagy and iron metabolism in patient-derived cells. The expression of ferritin and ferric iron increased and that of ferrous iron decreased in the patient cells with WDR45 variants. In addition, the expression of nuclear receptor coactivator 4 was markedly reduced in patient-derived cells. Furthermore, divalent metal transporter 1, which takes in ferrous iron, was upregulated, while ferroportin, which exports ferrous iron, was downregulated in patient-derived cells. The transfer of WDR45 via an adeno-associated virus vector restored WIPI4 and nuclear receptor coactivator 4 expression, reduced ferritin levels, and improved other phenotypes observed in patient-derived cells. As nuclear receptor coactivator 4 mediates the ferritin-specific autophagy, i.e., ferritinophagy, its deficiency impaired ferritinophagy, leading to the accumulation of ferric iron‒containing ferritin and insufficiency of ferrous iron. Because ferrous iron is required for various essential biochemical reactions, the changes in divalent metal transporter 1 and ferroportin levels may indicate a compensatory response for maintaining the intracellular levels of ferrous iron. Our study revealed that the pathophysiology of static encephalopathy of childhood with neurodegeneration in adulthood/β-propeller protein-associated neurodegeneration involves ferrous iron insufficiency via impaired ferritinophagy through nuclear receptor coactivator 4 expression reduction. Our findings could aid in developing a treatment strategy involving WDR45 manipulation, which may have clinical applications.

<title>Abstract</title><sec> <title>Background</title> Congenital central hypoventilation syndrome (CCHS) is a rare disease characterized by sleep apnea. Anoxia often occurs soon after birth, and it is important to prevent anoxia-mediated central nervous system complications; however, data on the relationship between respiratory management and the prognosis for intellectual development of patients with CCHS is not well yet investigate. </sec><sec> <title>Methods</title> We performed a retrospective chart review cohort study of patients with CCHS in Japan. We investigated the risk and prognostic factors for developmental outcomes and examined the disease in terms of its symptoms, diagnosis, complications, and treatment. </sec><sec> <title>Results</title> Of the 123 patients with CCHS included in the survey, 88 patients were 6 years old and older. They were divided into two group based on their intelligence quotient. Those treated using positive-pressure ventilation via tracheostomy in the first three months of life had a better developmental prognosis than those managed via tracheostomy after three months of age and those treated by ventilation using mask (OR = 3.80; 95% CI: 1.00–14.37, OR = 4.65; 95% CI: 1.11–19.37). There was no significant difference in physical development (<italic>P</italic> = 0.64). </sec><sec> <title>Conclusions</title> The best respiratory treatment for patients with CCHS is ventilation via tracheostomy, initiated ideally before the age of three months. </sec>

Niemann-Pick disease type C1 (NPC1) is a fatal congenital neurodegenerative disorder caused by mutations in the NPC1 gene, which is involved in cholesterol transport in lysosomes. Broad clinical manifestations of NPC1 include liver failure, pulmonary disorder, neurological deficits, and psychiatric symptoms. The main cause of death in NPC1 patients involves central nervous system (CNS) dysfunction; there is no essential treatment. We generated a tyrosine-mutant adeno-associated virus (AAV) 9/3 vector that expresses human NPC1 under a cytomegalovirus (CMV) promoter (AAV-CMV-hNPC1) and injected it into the left lateral ventricle (5 μL) and cisterna magna (10 μL) of Npc1 homo-knockout (Npc1-/-) mice. Each mouse received total 1.35 × 1011 vector genome on days 4 or 5 of life. AAV-treated Npc1-/- mice (n = 11) had an average survival of >28 weeks, while all saline-treated Npc1-/- mice (n = 11) and untreated Npc1-/- mice (n = 6) died within 16 weeks. Saline-treated and untreated Npc1-/- mice lost body weight from 7 weeks until death. However, the average body weight of AAV-treated Npc1-/- mice increased until 15 weeks. AAV-treated Npc1-/- mice also showed a significant improvement in the rotarod test performance. A pathological analysis at 11 weeks showed that cerebellar Purkinje cells were preserved in AAV-treated Npc1-/- mice. In contrast, untreated Npc1-/- mice showed an almost total loss of cerebellar Purkinje cells. Combined injection into both the lateral ventricle and cisterna magna achieved broader delivery of the vector to the CNS, leading to better outcomes than noted in previous reports, with injection into the lateral ventricles or veins alone. In AAV-treated Npc1-/- mice, vector genome DNA was detected widely in the CNS and liver. Human NPC1 RNA was detected in the brain, liver, lung, and heart. Accumulated unesterified cholesterol in the liver was reduced in the AAV-treated Npc1-/- mice. Our results suggest the feasibility of gene therapy for patients with NPC1.

Several patients with beta-propeller protein-associated neurodegeneration (BPAN)/static encephalopathy with neurodegeneration in adulthood have been reported to present Rett syndrome (RTT)-like features. This report presents an individual with BPAN showing clinical features of RTT. Psychomotor delay and epilepsy onset were noted at 1 year, and regression began at 4 years. Screening of the methyl-CpG binding protein 2 (MECP2) did not show variants. At 22 years, basal ganglia iron deposits were found on magnetic resonance imaging (MRI), and the WD-domain repeat 45 gene (WDR45) variant was identified. Review of the literature showed that BPAN with RTT-like features is associated with more epileptic seizures and less deceleration of head growth, breathing irregularities, and cold extremities than classic RTT with MECP2 variants. These clinical presentations may provide clues for differentiating between these two disorders. However, both WDR45 and MECP2 should be screened in patients presenting a clinical picture of RTT without specific MRI findings of BPAN.

OBJECTIVE: The early diagnosis of beta-propeller protein-associated neurodegeneration (BPAN) before distinct brain magnetic resonance imaging (MRI) findings of iron deposition occur remains challenging. This study examined whether children with BPAN have characteristic high-amplitude (>50 μV) fast activity (HAFA) on electroencephalography (EEG). METHODS: We conducted a retrospective analysis of EEG performed during childhood in five patients with BPAN. We also examined 143 EEGs from 59 patients with different etiologies, including epilepsy (n = 33), acute encephalopathy (n = 6), neurodevelopmental disorders (n = 5), non-epileptic events (n = 4), and others (n = 11). Trained electroencephalographers reviewed all of the EEGs. When excessive fast activity was observed, the amplitude, frequency, and locality were assessed. RESULTS: All five patients with BPAN underwent initial EEGs at 12-21 months old, and diffuse continuous HAFA (range 20-50 Hz) was observed on both awake and sleep EEGs. In the awake records, there was no clear posterior dominant rhythm in 4 of the 5 patients. Although 28% of the 143 EEGs had continuous excessive fast activity, mainly in the sleep records, only two (1.4%) exhibited HAFA when asleep, and their awake EEGs had clear posterior dominant rhythm. CONCLUSIONS: The EEGs of children with BPAN showed diffuse HAFA continuously when both awake and asleep, which is uncommon in children with other etiologies. SIGNIFICANCE: This study provides an important clue for the early diagnosis of BPAN.

<title>Abstract</title> Idiopathic restless legs syndrome (RLS) has a genetic basis wherein <italic>BTBD9</italic> is associated with a higher risk of RLS. Hemodialysis patients also exhibit higher rates of RLS compared with the healthy population. However, little is known about the relationship of <italic>BTBD9</italic> and end-stage renal disease to RLS pathophysiology. Here we evaluated sleep and leg muscle activity of <italic>Btbd9</italic> mutant (MT) mice after administration of serum from patients with either idiopathic or RLS due to end-stage renal disease (renal RLS) and investigated the efficacy of treatment with the dopamine agonist rotigotine. At baseline, the amount of rapid eye movement (REM) sleep was decreased and leg muscle activity during non-REM (NREM) sleep was increased in MT mice compared to wild-type (WT) mice. Wake-promoting effects of rotigotine were attenuated by injection of serum from RLS patients in both WT and MT mice. Leg muscle activity during NREM sleep was increased only in MT mice injected with serum from RLS patients of ideiopatic and renal RLS. Subsequent treatment with rotigotine ameliorated this altered leg muscle activity. Together these results support previous reports showing a relationship between the Btbd9/dopamine system and RLS, and elucidate in part the pathophysiology of RLS.

INTRODUCTION: Neuronal ceroid lipofuscinoses (NCLs; CLN) are mainly autosomal recessive neurodegenerative disorders characterized by the accumulation of autofluorescent lipopigments in neuronal and other cells. Symptoms include visual disabilities, motor decline, and epilepsy. Causative genes are CLN1, CLN2, CLN3, CLN5, CLN6, CLN7, CLN8, CLN10, CLN11, CLN12, CLN13, and CLN14. We present the fourth Japanese case with a CLN6 mutation. CASE PRESENTATION: At 3 years of age, our patient became clumsy and fell down easily. He developed focal seizures with impaired consciousness and was started on carbamazepine. He showed ataxic walking and dysarthria with increased deep tendon reflexes. Interictal electroencephalogram revealed slow waves in the left temporal and occipital areas. Brain magnetic resonance imaging showed cerebellar atrophy and ventriculomegaly. In optical coherence tomography (OCT), the inner layer of the retina was thick and highly reflective. Exome sequencing revealed a known homozygous mutation, C.794_976del, p. (Ser265del) in CLN6. DISCUSSION: A total of 130 cases of NCL with CLN6 mutations have been reported globally, of which only four were from Japan including the current patient. The deletion of serine at position 265 has been reported in six cases. Ser265 is located in a region of short repeated sequences that is susceptible to mutation. Clinical trials of gene therapy using adeno-associated virus serotype 9 have started for NCL6, making early diagnosis crucial. OCT examination might be helpful in achieving a diagnosis.

© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Genetic abnormalities in mitochondrial complex assembling factors are associated with leukoencephalopathy. We present a 1-year-old girl with consciousness disturbance after a respiratory infection. Brain MRI revealed leukoencephalopathy with bilaterally symmetrical hyperintensity in the substantia nigra, medial thalamic nuclei, and basal nuclei, as well as cavities in the cerebral white matter and corpus callosum. Lactate levels in the spinal fluid were high, while magnetic resonance spectroscopy of the cerebral white matter and basal nuclei showed high peak lactate levels, suggesting mitochondrial dysfunction. The respiratory enzyme activity of complex I was reduced to 17% to 21% in skeletal muscle. Whole exome sequencing identified compound heterozygous variations in NDUFAF3, involved in the assembly of mitochondrial complex I (c.342_343insGTG:p.117Valdup, c.505C > A:p.Pro169Thr). Two-dimensional, blue-native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE revealed reductions in Q-module (NDUFS2, NDUFS3, and NDUFA9) and P-module (NDUFB10 and NDUFB11) subunits, indicating disruption of mitochondrial complex I assembly. Our report expands the spectrum of clinical phenotypes associated with pathogenic variants of NDUFAF3.

Introduction: Levetiracetam has a high tolerability and is effective against various seizure types and epilepsy syndromes. However, no study has specifically evaluated the efficacy of levetiracetam in children with refractory epilepsy based on magnetic resonance imaging (MRI) findings and the presence of intellectual disability (ID). Methods: We retrospectively evaluated levetiracetam efficacy and safety in 49 pediatric patients who met the following inclusion criteria: (1) diagnosis of refractory epilepsy with first-line antiepileptic (AED) treatment &gt;= 2 years, (2) younger than 20 years old, and (3) received oral levetiracetam treatment for &gt;= 6 months. We assessed the relationships of these outcomes with MRI findings and ID status. Results: Eighteen (37%) patients achieved a &gt;= 50% reduction in seizure frequency, and the majority (78%) had no remarkable side effects. Twenty-two (45%) patients had previously been treated with more than seven antiepileptic drugs prior to levetiracetam. Among 18 patientg who achieved a 3 &gt;= 50% reduction in seizure frequency, 13 and 5 had negative and positive MRI findings, and 9 and 9 had and did not have ID, respectively. Conclusions: Our findings suggest that even for intractable pediatric cases with symptomatic etiology (i.e., MRI lesion and ID), levetiracetam has favorable efficacy for refractory epilepsy with tolerable adverse effects. (C) 2016 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

Joubert syndrome (JS) is rare recessive disorders characterized by the combination of hypoplasia/aplasia of the cerebellar vermis, thickened and elongated superior cerebellar peduncles, and a deep interpeduncular fossa which is defined by neuroimaging and is termed the molar tooth sign'. JS is genetically highly heterogeneous, with at least 29 disease genes being involved. To further understand the genetic causes of JS, we performed whole-exome sequencing in 24 newly recruited JS families. Together with six previously reported families, we identified causative mutations in 25 out of 30 (24+6) families (83.3%). We identified eight mutated genes in 27 (21+6) Japanese families, TMEM67 (7/27, 25.9%) and CEP290 (6/27, 22.2%) were the most commonly mutated. Interestingly, 9 of 12 CEP290 disease alleles were c.6012-12T&gt;A (75.0%), an allele that has not been reported in non-Japanese populations. Therefore c.6012-12T&gt;A is a common allele in the Japanese population. Importantly, one Japanese and one Omani families carried compound biallelic mutations in two distinct genes (TMEM67/RPGRIP1L and TMEM138/BBS1, respectively). BBS1 is the causative gene in Bardet-Biedl syndrome. These concomitant mutations led to severe and/or complex clinical features in the patients, suggesting combined effects of different mutant genes.

Static encephalopathy of childhood with neurodegeneration in adulthood (SENDA) is a recently established subtype of neurodegeneration with brain iron accumulation (NBIA)(1-3). By exome sequencing, we found de novo heterozygous mutations in WDR45 at Xp11.23 in two individuals with SENDA, and three additional WDR45 mutations were identified in three other subjects by Sanger sequencing. Using lymphoblastoid cell lines (LCLs) derived from the subjects, aberrant splicing was confirmed in two, and protein expression was observed to be severely impaired in all five. WDR45 encodes WD-repeat domain 45 (WDR45). WDR45 (also known as WIP14) is one of the four mammalian homologs of yeast Atgl 8, which has an important role in autophagy(4,5). Lower autophagic activity and accumulation of aberrant early autophagic structures were demonstrated in the LCLs of the affected subjects. These findings provide direct evidence that an autophagy defect is indeed associated with a neurodegenerative disorder in humans.

The molecular mechanisms of neuronal morphology and synaptic vesicle transport have been largely elusive, and only a few of the molecules involved in these processes have been identified. Here, we developed a novel morphology-based gene trap method, which is theoretically applicable to all cell lines, to easily and rapidly identify the responsible genes. Using this method, we selected several gene-trapped clones of rat pheochromocytoma PC12 cells, which displayed abnormal morphology and distribution of synaptic vesicle-like microvesicles (SLMVs). We identified several genes responsible for the phenotypes and analyzed three genes in more detail. The first gene was BTB/POZ domain-containing protein 9 (Btbd9), which is associated with restless legs syndrome. The second gene was cytokine receptor-like factor 3 (Crlf3), whose involvement in the nervous system remains unknown. The third gene was single-stranded DNA-binding protein 3 (Ssbp3), a gene known to regulate head morphogenesis. These results suggest that Btbd9, Crlf3, and Ssbp3 regulate neuronal morphology and the biogenesis/transport of synaptic vesicles. Because our novel morphology-based gene trap method is generally applicable, this method is promising for uncovering novel genes involved in the function of interest in any cell lines.-Hashimoto, Y., Muramatsu, K., Kunii, M., Yoshimura, S., Yamada, M., Sato, T., Ishida, Y., Harada, R., Harada, A. Uncovering genes required for neuronal morphology by morphology-based gene trap screening with a revertible retrovirus vector. FASEB J. 26, 4662-4674 (2012). www.fasebj.org

Delirious behavior associated with influenza usually has an onset within a few days after fever and lasts &lt;24 hours. As we encountered several patients with 2009 H1N1 influenza who presented with late-onset and long-standing delirious behavior, we retrospectively evaluated the clinical, radiologic, and laboratory features to elucidate the possible pathophysiology. This information was collected on 5 previously healthy patients (2 boys and 3 girls, aged 10-15 years) with 2009 H1N1 influenza who presented with late onset (&gt;3 days after fever) and long-standing (&gt;48 hours) delirious behavior. Each exhibited mild to moderate drowsiness between the episodes of delirious behavior. Electroencephalography was normal except for 1 patient with high voltage and slow activity bilaterally in the occipital regions. Brain MRI was normal. The outcome was excellent with no neurologic sequel in 4 of the 5 patients. In all 5 patients, autoantibodies against N-methyl-D-aspartate type glutamate receptor were elevated or positive in cerebrospinal fluid or serum; the autoantibody levels normalized in the 3 patients who had follow-up studies. This study indicates that 2009 H1N1 influenza has a tendency to cause late-onset and long-standing delirious behavior, at least in Japanese children. Mild autoimmune-mediated encephalitis should be considered as an underlying cause. Pediatrics 2012;129:e1068-e1071

To determine the neuronal function of genes in vivo, the neuron-specific deletion of a target gene in animals is required. Tau, a microtubule-associated protein, is expressed abundantly in neurons but scarcely in glias and other tissues. Therefore, to generate mice that express Cre recombinase in neurons, we inserted Cre recombinase into the tau locus. By crossing these tau-Cre mice with ROSA26 lacZ reporter mice, we observed Cre recombinase activity in the neurons from most of the central nervous system, but not in glias nor in non-neuronal tissues. This neuronal-specific activity appeared during embryogenesis. We further crossed tau-Cre mice with rab8 &apos;floxed&apos; mice, and showed that the recombination was nearly complete in the brain, but incomplete or non-detectable in other tissues. Thus, tau-Cre knockin mouse is a useful tool for studying the neuronal function of a gene in vivo. (c) 2008 Elsevier Inc. All rights reserved.

To investigate the neuronal function of genes in vivo, a neuron-specific and inducible gene targeting system is desirable. In this study, we generated a knockin mouse line that expresses a fusion protein consisting of the Cre recombinase and the progesterone receptor (CrePR) in neurons. The neuron-specific expression of CrePR was attained by inserting CrePR gene into the tau locus, because T is expressed strongly in neurons but scarcely in glias and other tissues. By crossing this knockin mouse line (tau(CrePR)) with ROSA26 lacZ reporter mouse line (R26R), we observed that the anti progesterone RU486 could induce recombinase activity of the CrePR specifically in neurons. Thus, tau(CrePR) knockin line is a useful tool for studying neuronal gene functions.