Overview: Research on the Genetic Architecture of the Developing Cerebral Cortex in Norms and Diseases.

The human brain is characterized by high cell numbers, diverse cell types with diverse functions, and intricate connectivity with an exceedingly broad surface of the cortex. Human-specific brain development was accomplished by a long timeline for maturation from the prenatal period to the third decade of life. The long timeline makes complicated architecture and circuits of human cerebral cortex possible, and it makes human brain vulnerable to intrinsic and extrinsic insults resulting in the development of variety of neuropsychiatric disorders. Unraveling the molecular and cellular processes underlying human brain development under the elaborate regulation of gene expression in a spatiotemporally specific manner, especially that of the cortex will provide a biological understanding of human cognition and behavior in health and diseases. Global research consortia and the advancing technologies in brain science including functional genomics equipped with emergent neuroinformatics such as single-cell multiomics, novel human models, and high-volume databases with high-throughput computation facilitate the biological understanding of the development of the human brain cortex. Knowing the process of interplay of the genome and the environment in cortex development will lead us to understand the human-specific cognitive function and its individual diversity. Thus, it is worthwhile to overview the recent progress in neurotechnology to foresee further understanding of the human brain and norms and diseases.

Association of moderately abnormal behavior and administered neuraminidase inhibitors.

Our earlier study investigated the incidence of severe abnormal behavior associated with neuraminidase inhibitors (NIs), but some studies have specifically examined the association of oseltamivir use and moderately abnormal behavior. Therefore, this study was undertaken to assess associations between moderately abnormal behavior and administered drugs. All cases of patients with influenza who exhibited moderately abnormal behavior were reported to us by physicians of all sentinel clinics and hospitals for influenza throughout Japan. Open Data of the National Database of Electronic Medical Claims include the numbers of patients diagnosed as having influenza who were prescribed NI. Incidence by NI was tested using Fisher's exact test. We received 518 moderately abnormal cases in 5-9-year-olds and 207 moderately abnormal behavior cases in 10-19-year-olds. The incidence among NI ranged from 193 per one million influenza patients in laninamivir among 10-19-year-olds to 1021 for peramivir among 5-9-year-olds. Estimation results revealed the order of risk among NIs as peramivir, oseltamivir, zanamivir and laninamivir in moderate abnormal behavior. Because of data limitations, risk among patients with and without NI cannot be compared.

Association of severe abnormal behavior and acetaminophen with/without neuraminidase inhibitors.

BACKGROUND: Even though abnormal behavior related with influenza and neuraminidase inhibitors (NI) has been discussed, the risks of acetaminophen and co-administration of NI and acetaminophen have not been examined. This study assesses those risks. MATERIALS AND METHODS: All cases of patients with influenza who present with severe abnormal behavior are reported by physicians of all clinics and hospitals throughout Japan. The numbers of people diagnosed as having influenza, whether prescribed NI and acetaminophen or not, were extracted from the National Database of Electronic Medical Claims (NDBEMC). The study period was from September 2009 to March 2016. RESULTS: We found two consistent results among four combinations of age class and severity. The one was that patients who did not use NI or acetaminophen showed significantly higher incidence of abnormal behavior than zanamivir with acetaminophen, another one was that patients with oseltamivir only has higher incidence than zanamivir with acetaminophen. Concerning about acetaminophen, the use of it significantly decrease risk for severe and the most severe instances in 5-9-year-old patients with laninamivir and the severe instances in 10-19-year-old patients with zanamivir. DISCUSSION: We also demonstrated that acetaminophen alone or co-administered with NI does not seem to raise the risk of abnormal behavior in influenza patients.

Severe abnormal behavior incidence after administration of neuraminidase inhibitors using the national database of medical claims.

An earlier study using the number of abnormal behaviors reported to the study group as the numerator and the number of influenza patient prescribed each neuraminidase inhibitor (NI) estimated by respective pharmaceutical companies found no significant difference among incidence rates of the most severe abnormal behaviors by type of NI throughout Japan. However, the dataset for the denominator used in that earlier study was the estimated number of prescriptions. In the present study, to compare the incidence rates of abnormal behavior more precisely among influenza patients administered several sorts of NI or administered no NI, we used data obtained from the National Database of Electronic Medical Claims (NDBEMC) as the denominator to reach a definitive conclusion. Results show that patients not administered any NI (hereinafter un-administered) or those administered peramivir sometimes showed higher risk of abnormal behavior than those administered oseltamivir, zanamivir, or laninamivir. However, the un-administered or peramivir patients were fewer than those taking other NI. Therefore, accumulation of data through continued research is expected to be necessary to reach a definitive conclusion about the relation between abnormal behavior and NI in influenza patients. Since severe abnormal behaviors with all types of NI or of un-administered patients have been reported, there are some risks in the administration of NI or even in un-administered cases. Therefore, we infer that the policy mandating package inserts in all types of NI.

Inquiry into some gap among oseltamivir use and severe abnormal behavior in Japanese children and adolescents with influenza.

The Fukushima research has examined data form a cohort study of 10,000 Japanese children under 18 years old with influenza during three months to demonstrate that the relative risk of A-type abnormal behavior of patients with oseltamivir was 30 times greater than without oseltamivir. By contrast, our research group found that patients who had been administered no neuraminidase inhibitors (NI) or those administered peramivir had higher risk of abnormal behavior than those administered oseltamivir, zanamivir, or laninamivir. A plausible explanation for this gap is that the two studies specifically examined different criteria to report abnormal behavior. In actually, some A-type abnormal behavior might not be life-threatening. Our definition of severe abnormal behavior is better matched to public health concerns and comparison among incidents according to the administered drug is more appropriate as an analytical procedure.

In vitro and in vivo evaluation of tissue-cultured mountain ginseng on penile erection.

BACKGROUND: Progressed tissue culture techniques have allowed us to easily obtain mass products of tissue-cultured mountain ginseng over 100 yr old (TCMG-100). We investigated the effects of TCMG-100 extract on erectile function using in vitro and in vivo studies. METHODS: To examine the relaxation effects and mechanisms of action of TCMG-100 on rabbit cavernosal strips evaluated in an organ bath. To investigate the long-term treatment effect of TCMG-100, 8-wk administration was performed. After administration of TCMG-100, intracavernosal pressure, cyclic guanosine monophosphate and nitric oxide (NO) levels of cavernosal tissue, serum testosterone level, histological observation of collagen fiber, endothelium, smooth muscle cell, and transforming growth factor-ß1 were investigated. RESULTS: TCMG-100 extract displayed dose-dependent relaxation effects on precontracted rabbit corporal smooth muscle. The TCMG-100-induced relaxation was significantly reduced by removing the endothelium, and treatment with an NO synthase inhibitor or NO scavenger. Eight weeks of TCMG-100 administration increased intracavernosal pressure in a rat model. The levels of cyclic guanosine monophosphate and NO in the corpus callosum and serum testosterone level were also increased by TCMG-100 treatment. Furthermore, histological evaluation of collagen, smooth muscle, and endothelium showed increases in endothelium and smooth muscle, and a decrease in transforming growth factor-ß1 expression. CONCLUSION: These relaxation effects on corporal smooth muscle and increased erectile function suggest that TCMG-100 might be used as an alternative herbal medicine to improve erectile function.

Role of Class III phosphoinositide 3-kinase in the brain development: possible involvement in specific learning disorders.

Class III phosphoinositide 3-kinase (PIK3C3 or mammalian vacuolar protein sorting 34 homolog, Vps34) regulates vesicular trafficking, autophagy, and nutrient sensing. Recently, we reported that PIK3C3 is expressed in mouse cerebral cortex throughout the developmental process, especially at early embryonic stage. We thus examined the role of PIK3C3 in the development of the mouse cerebral cortex. Acute silencing of PIK3C3 with in utero electroporation method caused positional defects of excitatory neurons during corticogenesis. Time-lapse imaging revealed that the abnormal positioning was at least partially because of the reduced migration velocity. When PIK3C3 was silenced in cortical neurons in one hemisphere, axon extension to the contralateral hemisphere was also delayed. These aberrant phenotypes were rescued by RNAi-resistant PIK3C3. Notably, knockdown of PIK3C3 did not affect the cell cycle of neuronal progenitors and stem cells at the ventricular zone. Taken together, PIK3C3 was thought to play a crucial role in corticogenesis through the regulation of excitatory neuron migration and axon extension. Meanwhile, when we performed comparative genomic hybridization on a patient with specific learning disorders, a 107 Kb-deletion was identified on 18q12.3 (nt. 39554147-39661206) that encompasses exons 5-23 of PIK3C3. Notably, the above aberrant migration and axon growth phenotypes were not rescued by the disease-related truncation mutant (172 amino acids) lacking the C-terminal kinase domain. Thus, functional defects of PIK3C3 might impair corticogenesis and relate to the pathophysiology of specific learning disorders and other neurodevelopmental disorders. Acute knockdown of Class III phosphoinositide 3-kinase (PIK3C3) evokes migration defects of excitatory neurons during corticogenesis. PIK3C3-knockdown also disrupts axon outgrowth, but not progenitor proliferation in vivo. Involvement of PIK3C3 in neurodevelopmental disorders might be an interesting future subject since a deletion mutation in PIK3C3 was detected in a patient with specific learning disorders (SLD).

Life-Threatening Abnormal Behavior Incidence in 10-19 Year Old Patients Administered Neuraminidase Inhibitors.

Much discussion has surrounded the association between the administration of neuraminidase inhibitors (NI) and severe abnormal behaviors, including sudden running away and jumping from a high place, which can be life-threatening if no one intervenes. Using data on the number of abnormal behaviors and patients who had been prescribed NI in Japan, we calculated the incidence rate of severe abnormal behaviors among influenza patients who had been prescribed NI. Then, we evaluated the relative risk between the four types of NI on severe abnormal behavior. We found no significant difference in the incidence rates of abnormal behavior by the type of NI. Results implicate that the current policy of package inserts, which warn physicians that patients who were administered ANY type of NI might exhibit abnormal behavior, seems to be appropriate.

CASPR2 forms a complex with GPR37 via MUPP1 but not with GPR37(R558Q), an autism spectrum disorder-related mutation.

Autism spectrum disorder (ASD) is a developmental brain disorder. Mutations in synaptic components including synaptic adhesion molecules have been found in ASD patients. Contactin-associated protein-like 2 (CASPR2) is one of the synaptic adhesion molecules associated with ASD. CASPR2 forms a complex with receptors via interaction with multiple PDZ domain protein 1 (MUPP1). Little is known about the relationship between impaired CASPR2-MUPP1-receptor complex and the pathogenesis of ASD. GPR37 is a receptor for survival factors. We recently identified mutations including R558Q in the G-protein-coupled receptor 37 (GPR37) gene in ASD patients. The mutated GPR37s accumulate in the endoplasmic reticulum. In this study, we show that GPR37 is a component of the CASPR2-MUPP1 receptor complex in the mouse brain. CASPR2 and GPR37 mainly interacted with the PDZ3 and PDZ11 domains of MUPP1, respectively. Compared to GPR37, GPR37(R558Q) slightly interacted with MUPP1 and caused dendritic alteration. GPR37, but not GPR37(R558Q) nor GPR37-deltaC which lacks its PDZ binding domain, was transported to the cell surface by MUPP1. In primary hippocampal neurons, GPR37 co-localized with MUPP1 and CASPR2 at the synapse, but not GPR37(R558Q). Thus, ASD-related mutation of GPR37 may cause the impaired CASPR2-MUPP1-GPR37 complex on the dendrites associated with one of the pathogenesis of ASD. In this study, we identified that GPR37 is a component of the MUPP1 and CASPR2 receptor complex. Autism deleterious mutated GPR37(R558Q) slightly interacts with MUPP1 and retains in ER, resulting in dendritic alteration. In neuron, GPR37, but not GPR37(R558Q), is transported to the dendrite and synapse by MUPP1. Thus, ASD-related mutation of GPR37 may cause the impaired CASPR2-MUPP1-GPR37 complex on the dendrites associated with one of the pathogenesis of ASD.

The association of GPR85 with PSD-95-neuroligin complex and autism spectrum disorder: a molecular analysis.

BACKGROUND: Autism spectrum disorder (ASD) has a complex genetic etiology. Some symptoms and mutated genes, including neuroligin (NLGN), neurexin (NRXN), and SH3 and multiple ankyrin repeat domains protein (SHANK), are shared by schizophrenia and ASD. Little is known about the molecular pathogenesis of ASD. One of the possible molecular pathogenesis is an imbalance of excitatory and inhibitory receptors linked with the NLGN-PSD-95-SHANK complex via postsynaptic density protein/Drosophila disc large tumor suppressor/zonula occludens-1 protein (PDZ) binding. In the present study, we focused on GPR85 as a candidate gene for ASD because the C-terminal amino acid sequence of GPR85 [Thr-Cys-Val-Ile (YCVI)] is classified as a type II PDZ-binding motif, and GPR85 is a risk factor for schizophrenia. GPR85 is an orphan receptor that regulates neural and synaptic plasticity and modulates diverse behaviors, including learning and memory. While searching for molecules that associate with GPR85, we found that GPR85 was associated with postsynaptic density protein (PSD)-95 linked with NLGN in the brain. METHODS: We examined the proteins that associate with the C-terminal sequence of GPR85 by pull-down assay and immunoblot analysis and searched for a mutation of the GPR85 gene in patients with ASD. We used immunostaining to examine the intracellular localization of mutated GPR85 and its influence on the morphology of cells and neurons. RESULTS: The C-terminal sequence of GPR85 interacted with PSD-95 at PDZ1, while NLGN interacted with PSD-95 at PDZ3. Two male patients with ASD from independent Japanese families possessed inherited missense mutations at conserved sites in GPR85: one had T1033C (M152T) and the other had G1239T (V221L). These mutations were located in a domain related to G protein interaction and signal transduction. In contrast to wild-type GPR85, mutated GPR85 was more preferentially accumulated, causing endoplasmic reticulum stress, and disturbed the dendrite formation of hippocampal neurons. CONCLUSIONS: GPR85 associated with the PSD-95 linked with NLGN, which is related to ASD. GPR85 carrying the mutations detected in ASD patients disturbed dendrite formation that could be the candidate for molecular pathogenesis of ASD through the associated NLGN-PSD-95 receptor complex.

Role of an adaptor protein Lin-7B in brain development: possible involvement in autism spectrum disorders.

Using comparative genomic hybridization analysis for an autism spectrum disorder (ASD) patient, a 73-Kb duplication at 19q13.33 (nt. 49 562 755-49 635 956) including LIN7B and 5 other genes was detected. We then identified a novel frameshift mutation in LIN7B in another ASD patient. Since LIN7B encodes a scaffold protein essential for neuronal function, we analyzed the role of Lin-7B in the development of cerebral cortex. Acute knockdown of Lin-7B with in utero electroporation caused a delay in neuronal migration during corticogenesis. When Lin-7B was knocked down in cortical neurons in one hemisphere, their axons failed to extend efficiently into the contralateral hemisphere after leaving the corpus callosum. Meanwhile, enhanced expression of Lin-7B had no effects on both cortical neuron migration and axon growth. Notably, silencing of Lin-7B did not affect the proliferation of neuronal progenitors and stem cells. Taken together, Lin-7B was found to play a pivotal role in corticogenesis through the regulation of excitatory neuron migration and interhemispheric axon growth, while further analyses are required to directly link functional defects of Lin-7B to ASD pathophysiology. Lin-7 plays a pivotal role as a scaffold protein in synaptic development and plasticity. Based on genetic analyses we identified mutations in LIN-7B gene in some ASD (autism-spectrum disorder) patients. Functional defects in Lin-7B caused abnormal neuronal migration and interhemispheric axon growth during mouse brain development. Thus, functional deficiency in Lin-7B could be implicated in clinical phenotypes in some ASD patients through bringing about abnormal cortical architecture.

Apolipoprotein AII levels are associated with the UP/UCr levels in idiopathic steroid-sensitive nephrotic syndrome.

BACKGROUND: Various humoral factors have been proposed as causal agents of idiopathic steroid-sensitive nephrotic syndrome (ISSNS), resulting in varying data. We used mass spectrometry (MS) to analyze serum proteins in a search for proteins that might be involved in ISSNS pathophysiology. METHODS: Serial serum samples were obtained from 33 children with ISSNS. Samples were collected during Phase A1 [the acute phase prior to steroid treatment (STx)], Phase A2 (remission with STx), and Phase A3 (remission without any medication). We also included age- and sex-matched two control groups comprising children with normal urinalysis (Group B) and children with a nephrotic syndrome other than ISSNS (Group C). The urinary protein/urinary creatinine (UP/UCr) ratios were not statistically different between Phase A1 and Group C. Samples were analyzed using surface-enhanced laser desorption/ionization time of flight MS. RESULTS: A total of 207 peptide ion peaks were detected in the range of m/z 2000-10000. Four peptide ions (m/z 6444, 6626, 8695, and 8915) were detected at significant elevation during Phase A1 compared with Phase A2, Phase A3, and Group C. The intensities of m/z 6444 and 8695 were higher in Phase A3 than in Group B. There were significant correlations between the intensities of m/z 6626, 8695, and 8915 and UP/UCr levels. The m/z 8695 was identified as apolipoprotein AII. CONCLUSIONS: Apolipoprotein AII was detected as a protein associated with the UP/UCr levels in pediatric ISSNS. Our findings present an interesting starting point for further investigation into the pathophysiology of ISSNS.

Acute neuropharmacological effects of atomoxetine on inhibitory control in ADHD children: a fNIRS study.

The object of the current study is to explore the neural substrate for effects of atomoxetine (ATX) on inhibitory control in school-aged children with attention deficit hyperactivity disorder (ADHD) using functional near-infrared spectroscopy (fNIRS). We monitored the oxy-hemoglobin signal changes of sixteen ADHD children (6-14 years old) performing a go/no-go task before and 1.5 h after ATX or placebo administration, in a randomized, double-blind, placebo-controlled, crossover design. Sixteen age- and gender-matched normal controls without ATX administration were also monitored. In the control subjects, the go/no-go task recruited the right inferior and middle prefrontal gyri (IFG/MFG), and this activation was absent in pre-medicated ADHD children. The reduction of right IFG/MFG activation was acutely normalized after ATX administration but not placebo administration in ADHD children. These results are reminiscent of the neuropharmacological effects of methylphenidate to up-regulate reduced right IFG/MFG function in ADHD children during inhibitory tasks. As with methylphenidate, activation in the IFG/MFG could serve as an objective neuro-functional biomarker to indicate the effects of ATX on inhibitory control in ADHD children. This promising technique will enhance early clinical diagnosis and treatment of ADHD in children, especially in those with a hyperactivity/impulsivity phenotype.

Abnormal behavior during influenza in Japan during the last seven seasons: 2006-2007 to 2012-2013.

A few mortalities and cases of severe abnormal behavior have been reported after oseltamivir administration for influenza, thus increasing medical and public concerns regarding the drug's safety. We investigated the association between oseltamivir and abnormal behavior for seven years. All outpatient clinics and hospitals all over the country were requested to report severe abnormal behavior that could have resulted in a fatality if nobody intervened, such as abrupt running outside the home or intention of jumping off a building. The survey was performed prospectively between the 2007-2008 and 2012-2013 seasons, and retrospectively for the 2006-2007 season. As the result of the investigation, eight-hundred fifty-eight cases were reported and among of them 95.7% were positive by the influenza rapid diagnosis test. The epidemic curve of severe abnormal behavior showed a pattern similar to influenza-like illness. The same pattern was observed regardless of age group, gender, or timing of the incidents after waking. Consequently, specific association between the types of medications used or the types of antiviral and abnormal behavior was not observed clearly. The reported abnormal behaviors include fatal cases that would have died if nobody had stopped. This suggested that patients with influenza should be observed with caution for possible abnormal behavior whether taking oseltamivir or other neuraminidase inhibitor anti-influenza drugs.

Utility of non-enhanced magnetic resonance imaging to detect acute pyelonephritis.

It has been established that enhanced computed tomography (CT) and (99m) Tc-dimercaptosuccinic acid renal scintigraphy ((99m) Tc-DMSA scintigraphy) used in conjunction with single-photon emission CT is a useful tool for the diagnosis of acute pyelonephritis (APN). The utility of non-enhanced magnetic resonance imaging (MRI), however, has not been investigated extensively for the diagnosis of APN or renal abscess in children. We describe the case of a 23-month-old boy with suspected APN who received non-enhanced MRI. Whole body diffusion-weighted imaging (DWI) was used, and a background body-signal suppression sequence was applied. High-intensity focal lesions were identified on DWI and low-intensity lesions on the apparent diffusion coefficient map in the acute phase. This case suggested that non-enhanced MRI could be a useful tool for the diagnosis of APN in children, because it can avoid the risks of not only radiation exposure but also nephrogenic systemic fibrosis associated with gadolinium-based contrast agents, especially in infants.

Persistent presence of the anti-myelin oligodendrocyte glycoprotein autoantibody in a pediatric case of acute disseminated encephalomyelitis followed by optic neuritis.

We report the case of a 5-year-old Japanese girl who initially had acute disseminated encephalomyelitis (ADEM) and was positive for the myelin oligodendrocyte glycoprotein (MOG) antibodies and developed unilateral optic neuritis (ON) 71 days after ADEM onset. The patient's serum was positive for the anti-MOG antibodies from the onset of ADEM to the development of ON. This phenotype has been reported in only two previous articles, and the specific mechanism of action of the anti-MOG antibodies is not yet understood. Our case suggests that the anti-MOG antibody can be associated with the pathogenesis of ADEM followed by ON. Thus, patients with ADEM who test positive for the anti-MOG antibody may be at risk of developing subsequent ON.

LIN7A depletion disrupts cerebral cortex development, contributing to intellectual disability in 12q21-deletion syndrome.

Interstitial deletion of 12q21 has been reported in four cases, which share several common clinical features, including intellectual disability (ID), low-set ears, and minor cardiac abnormalities. Comparative genomic hybridization (CGH) analysis using the Agilent Human Genome CGH 180K array was performed with the genomic DNA from a two-year-old Japanese boy with these symptoms, as well as hypoplasia of the corpus callosum. Consequently, a 14 Mb deletion at 12q21.2-q21.33 (nt. 77 203 574-91 264 613 bp), which includes 72 genes, was detected. Of these, we focused on LIN7A, which encodes a scaffold protein that is important for synaptic function, as a possible responsible gene for ID, and we analyzed its role in cerebral cortex development. Western blotting analyses revealed that Lin-7A is expressed on embryonic day (E) 13.5, and gradually increases in the mouse brain during the embryonic stage. Biochemical fractionation resulted in the enrichment of Lin-7A in the presynaptic fraction. Suppression of Lin-7A expression by RNAi, using in utero electroporation on E14.5, delayed neuronal migration on postnatal day (P) 2, and Lin-7A-deficient neurons remained in the lower zone of the cortical plate and the intermediate zone. In addition, when Lin-7A was silenced in cortical neurons in one hemisphere, axonal growth in the contralateral hemisphere was delayed; development of these neurons was disrupted such that one half did not extend into the contralateral hemisphere after leaving the corpus callosum. Taken together, LIN7A is a candidate gene responsible for 12q21-deletion syndrome, and abnormal neuronal migration and interhemispheric axon development may contribute to ID and corpus callosum hypoplasia, respectively.

MAOA/B deletion syndrome in male siblings with severe developmental delay and sudden loss of muscle tonus.

Deletion of the monoamine oxidase (MAO)-A and MAO-B was detected in two male siblings and in their mother. The approximately 800-kb deletion, extending from about 43.0MB to 43.8MB, was detected by array comparative genomic hybridization analysis. The MAOA and MAOB genes were included in the deletion, but the adjacent Norrie disease gene, NDP, was not deleted. The boys had short stature, hypotonia, severe developmental delays, episodes of sudden loss of muscle tone, exiting behavior, lip-smacking and autistic features. The serotonin levels in their cerebrospinal fluid were extremely elevated. Another set of siblings with this deletion was reported previously. We propose recognition of MAOA/B deletion syndrome as a distinct disorder.