永井 拓

Successful completion of daily activities relies on the ability to select the relevant features of the environment for memory and recall. Disruption to these processes can lead to various disorders, such as attention-deficit hyperactivity disorder (ADHD). Dopamine is a neurotransmitter implicated in the regulation of several processes, including attention. In addition to the higher-order brain function, dopamine is implicated in the regulation of adult neurogenesis. Previously, we generated mice lacking Shati, an N-acetyltransferase-8-like protein on a C57BL/6J genetic background (Shati/Nat8l-/- ). These mice showed a series of changes in the dopamine system and ADHD-like behavioral phenotypes. Therefore, we hypothesized that deficiency of Shati/Nat8l would affect neurogenesis and attentional behavior in mice. We found aberrant morphology of neurons and impaired neurogenesis in the dentate gyrus of Shati/Nat8l-/- mice. Additionally, research has suggested that impaired neurogenesis might be because of the reduction of dopamine in the hippocampus. Galantamine (GAL) attenuated the attentional impairment observed in the object-based attention test via increasing the dopamine release in the hippocampus of Shati/Nat8l-/- mice. The α7 nicotinic acetylcholine receptor antagonist, methyllycaconitine, and dopamine D1 receptor antagonist, SCH23390, blocked the ameliorating effect of GAL on attentional impairment in Shati/Nat8l-/- mice. These results suggest that the ameliorating effect of GAL on Shati/Nat8l-/- attentional impairment is associated with activation of D1 receptors following increased dopamine release in the hippocampus via α7 nicotinic acetylcholine receptor. In summary, Shati/Nat8l is important in both morphogenesis and neurogenesis in the dentate gyrus and attention, possible via modulation of dopaminergic transmission.

BACKGROUND: Postpartum depression (PPD) is a major depressive disorder that occurs after childbirth. Objective diagnostic and predictive methods for PPD are important for early detection and appropriate intervention. DNA methylation has been recognized as a potential biomarker for major depressive disorder. In this study, we used methylation analysis and peripheral blood to search for biomarkers that could to lead to the development a predictive method for PPD. METHODS: Study participants included 36 pregnant women (18 cases and 18 controls determined after childbirth). Genome-wide DNA methylation profiles were obtained by analysis with an Infinium Human Methylation 450BeadChip. The association of DNA methylation status at each DNA methylation site with PPD was assessed using linear regression analysis. We also conducted functional enrichment analysis of PPD using The Database for Annotation, Visualization and Integrated Discovery 6.8 to explore enriched functional-related gene groups for PPD. RESULTS: In the analysis with postpartum depressed state as an independent variable, the difference in methylation frequency between the postpartum non-depressed group and the postpartum depressed group was small, and sites with genome-wide significant differences were not confirmed. After analysis by The Database for Annotation, Visualization and Integrated Discovery 6.8, we revealed four gene ontology terms, including axon guidance, related to postpartum depression. CONCLUSIONS: These findings may help with the development of an objective predictive method for PPD.

Although a number of studies have identified several convincing candidate genes or molecules, the pathophysiology of schizophrenia (SCZ) has not been completely elucidated. Therapeutic optimization based on pathophysiology should be performed as early as possible to improve functional outcomes and prognosis; to detect useful biomarkers for SCZ, which reflect pathophysiology and can be utilized for timely diagnosis and effective therapy. To explore biomarkers for SCZ, we employed fluorescence two-dimensional differential gel electrophoresis (2D-DIGE) of lymphoblastoid cell lines (LCLs) (1st sample set: 30 SCZ and 30 CON). Differentially expressed proteins were sequenced by liquid chromatography tandem-mass spectrometry (LC-MS/MS) and identified proteins were confirmed by western blotting (WB) (1st and 2nd sample set: 60 SCZ and 60 CON). Multivariate logistic regression analysis was performed to identify an optimal combination of biomarkers to create a prediction model for SCZ. Twenty protein spots were differentially expressed between SCZ and CON in 2D-DIGE analysis and 22 unique proteins were identified by LC-MS/MS. Differential expression of eight of 22 proteins was confirmed by WB. Among the eight candidate proteins (HSPA4L, MX1, GLRX3, UROD, MAPRE1, TBCB, IGHM, and GART), we successfully constructed logistic regression models comprised of 4- and 6-markers with good discriminative ability between SCZ and CON. In both WB and gene expression analysis of LCL, MX1 showed reproducibly significant associations. Moreover, Mx1 and its related proinflamatory genes (Mx2, Il1b, and Tnf) were also up-regulated in poly I:C-treated mice. Differentially expressed proteins might be associated with molecular pathophysiology of SCZ, including dysregulation of immunological reactions and potentially provide diagnostic and prognostic biomarkers.

主要組織適合遺伝子複合体クラスI(major histocompatibility complex class I;MHCI)は中枢神経系において主に神経細胞に発現し、スパインの刈り込みなどに関与している。グリア細胞におけるMHCIの病態生理学的役割については不明な点が多いことから、我々は末梢性免疫炎症反応による中枢神経系におけるMHCIの発現変化、ならびに前頭前皮質のアストロサイトにおける膜貫通型MHCI/H-2Dあるいは分泌型MHCI/sH-2Dの発現が周囲の細胞と高次脳機能に及ぼす影響に着目して研究を行ってきた。成熟マウスにpolyriboinosinic-polyribocytidilic acid(polyI:C)を腹腔内投与あるいはIFN-γ遺伝子を尾静脈から導入し、大脳皮質を摘出してMHCIのmRNA量を測定した。コントロール群と比較して、polyI:C処置群では、MHCIのmRNAレベルが有意に上昇し、同様の結果がIFN-γ遺伝子導入マウスでも認められた。MHCIのmRNA発現については神経細胞およびアストロサイトで増加することをin situ hybridization法により確認した。H-2DあるいはsH-2D遺伝子を導入したアストロサイトにおいて、同蛋白質は主にエキソソームに局在し、細胞外へ分泌されることが示唆された。アデノ随伴ウイルスベクターを用いてアストロサイト特異的にH-2DあるいはsH-2Dを前頭前皮質に発現させたマウスではミクログリアの活性化、パルブアルブミン陽性細胞数の減少、スパイン密度の低下などが認められ、社会性行動と物体認知記憶の障害が観察された。これらの障害はエキソソーム合成阻害剤GW4869により改善したことから、アストロサイトにおけるMHCIはエキソソームを介して近傍の細胞に影響し脳機能障害を惹起することが示唆された。(著者抄録)

Medium spiny neurons (MSNs) expressing dopamine D1 receptor (D1R) or D2 receptor (D2R) are major components of the striatum. Stimulation of D1R activates protein kinase A (PKA) through Golf to increase neuronal activity, while D2R stimulation inhibits PKA through Gi. Adenosine A2A receptor (A2AR) coupled to Golf is highly expressed in D2R-MSNs within the striatum. However, how dopamine and adenosine co-operatively regulate PKA activity remains largely unknown. Here, we measured Rap1gap serine 563 phosphorylation to monitor PKA activity and examined dopamine and adenosine signals in MSNs. We found that a D1R agonist increased Rap1gap phosphorylation in striatal slices and in D1R-MSNs in vivo. A2AR agonist CGS21680 increased Rap1gap phosphorylation, and pretreatment with the D2R agonist quinpirole blocked this effect in striatal slices. D2R antagonist eticlopride increased Rap1gap phosphorylation in D2R-MSNs in vivo, and the effect of eticlopride was blocked by the pretreatment with the A2AR antagonist SCH58261. These results suggest that adenosine positively regulates PKA in D2R-MSNs through A2AR, while this effect is blocked by basal dopamine in vivo. Incorporating computational model analysis, we propose that the shift from D1R-MSNs to D2R-MSNs or vice versa appears to depend predominantly on a change in dopamine concentration.

Medium spiny neurons (MSN) in the nucleus accumbens (NAc) are a fundamental component of various aspects of motivated behavior. Although mitogen-activated protein kinase (MAPK) signaling plays a crucial role in several types of learning, the cell type-specific role of MAPK pathway in stimulus-reward learning and motivation remains unclear. We herein investigated the role of MAPK in accumbal MSNs in reward-associated learning and memory. During the acquisition of Pavlovian conditioning, the number of phosphorylated MAPK1/3-positive cells was increased significantly and exclusively in the NAc core by 7-days of extensive training. MAPK signaling in the respective D1R- and D2R-MSNs was manipulated by transfecting an adeno-associated virus (AAV) plasmid into the NAc of Drd1a-Cre and Drd2-Cre transgenic mice. Potentiation of MAPK signaling shifted the learning curve of Pavlovian conditioning to the left only in Drd1a-Cre mice, whereas such manipulation in D2R-MSNs had negligible effects. In contrast, MAPK manipulation in D2R-MSNs of the NAc core significantly increased motivation for food rewards as found in Drd1a-Cre mice. These results suggest that MAPK signaling in the D1R-MSNs of NAc core plays an important role in stimulus-reward learning, while MAPK signaling in both D1R- and D2R-MSNs is involved in motivation for natural rewards.

Reelin protein (RELN), an extracellular matrix protein, plays multiple roles that range from embryonic neuronal migration to spine formation in the adult brain. Results from genetic studies have suggested that RELN is associated with the risk of psychiatric disorders, including schizophrenia (SCZ). We previously identified a novel exonic deletion of RELN in a patient with SCZ. High-resolution copy number variation analysis revealed that this deletion included exons 52 to 58, which truncated the RELN in a similar manner to the Reln Orleans mutation (Relnrl-Orl). We examined the clinical features of this patient and confirmed a decreased serum level of RELN. To elucidate the pathophysiological role of the exonic deletion of RELN in SCZ, we conducted behavioral and neurochemical analyses using heterozygous Relnrl-Orl/+ mice. These mice exhibited abnormalities in anxiety, social behavior, and motor learning; the deficits in motor learning were ameliorated by antipsychotics. Methamphetamine-induced hyperactivity and dopamine release were significantly reduced in the Relnrl-Orl/+ mice. In addition, the levels of GABAergic markers were decreased in the brain of these mice. Taken together, our results suggest that the exonic deletion of RELN plays a pathological role, implicating functional changes in the dopaminergic and GABAergic systems, in the pathophysiology of SCZ.

In the central nervous system, major histocompatibility complex class I (MHCI) molecules are mainly expressed in neurons, and neuronal MHCI have roles in synapse elimination and plasticity. However, the pathophysiological significance of astroglial MHCI remains unclear. We herein demonstrate that MHCI expression is up-regulated in astrocytes in the medial prefrontal cortex (mPFC) following systemic immune activation by an intraperitoneal injection of polyinosinic-polycytidylic acid (polyI:C) or hydrodynamic interferon (IFN)-γ gene delivery in male C57/BL6J mice. In cultured astrocytes, MHCI/H-2D largely co-localized with exosomes. To investigate the role of astroglial MHCI, H-2D, or sH-2D was expressed in the mPFC of male C57/BL6J mice using an adeno-associated virus vector under the control of a glial fibrillary acidic protein promoter. The expression of astroglial MHCI in the mPFC impaired sociability and recognition memory in mice. Regarding neuropathological changes, MHCI expression in astrocytes significantly activated microglial cells, decreased parvalbumin-positive cell numbers, and reduced dendritic spine density in the mPFC. A treatment with GW4869 that impairs exosome synthesis ameliorated these behavioral and neuropathological changes. These results suggest that the overexpression of MHCI in astrocytes affects microglial proliferation as well as neuronal numbers and spine densities, thereby leading to social and cognitive deficits in mice, possibly via exosomes created by astrocytes.

Neuronal intrinsic homeostatic scaling-down of excitatory synapse has been implicated in epilepsy pathogenesis to prevent the neuronal circuits from hyperexcitability. Recent findings suggest a role for neuronal PAS domain protein 4 (Npas4), an activity-dependent neuron-specific transcription factor in epileptogenesis, however, the underlying mechanism by which Npas4 regulates epilepsy remains unclear. We herein propose that limbic seizure activity up-regulates Npas4-homer1a signaling in the hippocampus, thereby contributing to epileptogenesis in mice. The expression level of Npas4mRNA was significantly increased after the pentylenetetrazol (PTZ) treatment. Npas4KO mice developed kindling more rapidly than their wild-type littermates. The expression of Homer1a in the hippocampus increased after seizure activity. Npas4 increased Homer1a promoter activity in COS7 cells. The PTZ-stimulated induction of Homer1a was attenuated in the hippocampus of Npas4KO mice. The combination of fluorescence in situ hybridization and immunohistochemical analyses revealed that Homer1amRNA co-localized with the Npas4 protein after the convulsive seizure response. PTZ reduced excitatory synaptic transmission at the associational/commissural fibers-CA3 synapses through the Npas4-mediated down-regulation of postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in hippocampal CA3 neurons. The adeno-associated virus (AAV)-mediated expression of Homer1a resulted in lower α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptor GluA1 subunit levels in the hippocampal plasma membrane fraction than in that from AAV-EGFP-transfected Npas4KO mice. The development of kindling was more strongly suppressed in AAV-Homer1a-microinjected Npas4KO mice than in AAV-EGFP-microinjected Npas4KO mice. These results indicate that Npas4 functions as a molecular switch to initiate homeostatic scaling and the targeting of Npas4-Homer1a signaling may provide new approaches for the treatment of epilepsy. (Figure presented.).

Scaffold proteins play a pivotal role in making protein complexes, and organize binding partners into a functional unit to enhance specific signaling pathways. IQ motif-containing GTPase activating protein 1 (IQGAP1) is an essential protein for spine formation due to its role in scaffolding multiple signal complexes. However, it remains unclear how IQGAP1 interacts within the brain. In the present study, we screened novel IQGAP1-interacting proteins by a proteomic approach. As a novel IQGAP1-interacting protein, we identified valosin-containing protein (VCP) which is a causative gene in patients with inclusion body myopathy with Paget's disease of bone and frontotemporal dementia (IBMPFD). The physiological interaction of IQGAP1 with VCP was confirmed by an immunoprecipitation assay. Both the N-terminal (N-half) and C-terminal (C-half) fragments of IQGAP1 interacted with the N-terminal region of VCP. Co-localization of IQGAP1 and VCP was observed in the growth corn, axonal shaft, cell body, and dendrites in cultured hippocampal neurons at 4 days in vitro (DIV4). In cultured neurons at DIV14, IQGAP1 co-localized with VCP in dendrites. When HEK293T cells were co-transfected with IQGAP1 and VCP, an immunoprecipitation assay revealed that binding of IQGAP1 with disease-related mutant (R155H or A232E) VCP was markedly reduced compared to wild-type (WT) VCP. These results suggest that reduction of IQGAP1 and VCP interaction may be associated with the pathophysiology of IBMPFD.

Diphtheria toxin (DT) administration into trans genic mice that express the DT receptor (DTR) under control of specific promoters is often used for cell ablation studies in vivo. Because DTR is not expressed in mice, DT injection has been assumed to be nontoxic to cells in vivo. In this study, we demonstrated that DT application during the juvenile stage leads to hearing loss in wild-type mice. Auditory brainstem response measurement showed severe hearing loss in C57BL/6 mice administered DT during the juvenile period, and the hearing loss persisted into adulthood. However, ototoxicity did not occur when DT was applied on postnatal day 28 or later. Histological studies demonstrated that hearing loss was accompanied by signif-icant degeneration of inner and outer hair cells (HCs), as well as spiral ganglion neurons. Scanning electron microscopy showed quick degeneration of inner HCs within 3 days and gradual degeneration of outer HCs within 1 week. These results demonstrated that DT has ototoxic action on C57BL/6 mice during the juvenile period, but not thereafter, and the hearing loss was due to degeneration of inner and outer HCs by unknown DT-related mechanisms. (C) 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

Postoperative cognitive dysfunction (POCD) occurs in nearly one-third of patients after non-cardiac surgery. Many animal behavior studies have investigated the effect of general anesthesia on cognitive function. However, there have been no studies examining the effects on working memory specifically, with a focus on the retention of working memory. We demonstrate here that isoflurane anesthesia induces deficits in the retention of spatial working memory in rats, as revealed by an increase in isoflurane-induced across-phase errors in the delayed spatial win-shift (SWSh) task with a 30-min delay in an 8-arm radial arm maze on post-anesthesia days (PADs) 1,2,4, and 10. A post-hoc analysis revealed a significant increase in across-phase errors on PAD 1 and recovery on PAD 10 in the isoflurane group. In contrast, within-phase errors independent of the retention of working memory were unaffected by isoflurane. These results demonstrate that isoflurane anesthesia transiently impairs the retention of spatial working memory in rats.

Dopamine signaling in the brain is a complex phenomenon that strongly contributes to emotional behaviors. Medium spiny neurons (MSNs) play a major role in dopamine signaling through dopamine D1 receptors (D1Rs) or dopamine D2 receptors (D2Rs) in the striatum. cAMP/protein kinase A (PKA) regulates phosphorylation signals downstream of D1Rs, which affects the excitability of MSNs, leading to reward-associated emotional expression and memory formation. A combination of phosphoproteomic approaches and the curated KANPHOS database can be used to elucidate the physiological and pathophysiological functions of dopamine signaling and other monoamines. Emerging evidence from these techniques suggests that the Rap1 pathway plays a crucial role in the excitability of MSNs, leading to the expression of emotional behaviors.

Dopamine (DA) type 1 receptor (D1R) signaling in the striatum presumably regulates neuronal excitability and reward-related behaviors through PKA. However, whether and how D1Rs and PKA regulate neuronal excitability and behavior remain largely unknown. Here, we developed a phosphoproteomic analysis method to identify known and novel PKA substrates downstream of the D1R and obtained more than 100 candidate substrates, including Rap1 GEF (Rasgrp2). We found that PKA phosphorylation of Rasgrp2 activated its guanine nucleotide-exchange activity on Rap1. Cocaine exposure activated Rap1 in the nucleus accumbens in mice. The expression of constitutively active PKA or Rap1 in accumbal D1R-expressing medium spiny neurons (D1R-MSNs) enhanced neuronal firing rates and behavioral responses to cocaine exposure through MAPK. Knockout of Rap1 in the accumbal D1R-MSNs was sufficient to decrease these phenotypes. These findings demonstrate a novel DA-PKA-Rap1-MAPK intracellular signaling mechanism in D1R-MSNs that increases neuronal excitability to enhance reward-related behaviors.

Neuronal circuit plasticity during development is fundamental for precise network formation. Pioneering studies of the developmental visual cortex indicated that noradrenaline (NA) is crucial for ocular dominance plasticity during the critical period in the visual cortex. Recent research demonstrated tonotopic map formation by NA during the critical period in the auditory system, indicating that NA also contributes to synaptic plasticity in this system. The lateral superior olive (LSO) in the auditory system receives glutamatergic input from the ventral cochlear nucleus (VCN) and undergoes circuit remodeling during postnatal development. LSO is innervated by noradrenergic afferents and is therefore a suitable model to study the function of NA in refinement of neuronal circuits. Chemical lesions of the noradrenergic system and chronic inhibition of α2-adrenoceptors in vivo during postnatal development in mice disrupted functional elimination and strengthening of VCN-LSO afferents. This was potentially mediated by activation of presynaptic α2-adrenoceptors and inhibition of glutamate release because NA presynaptically suppressed excitatory postsynaptic current (EPSC) through α2-adrenoceptors during the first two postnatal weeks in an in vitro study. Furthermore, NA and α2-adrenoceptor agonist induced long-term suppression of EPSCs and decreased glutamate release. These results suggest that NA has a critical role in synaptic refinement of the VCN-LSO glutamatergic pathway through failure of synaptic transmission. Because of the ubiquitous distribution of NA afferents and the extensive expression of α2-adrenoceptors throughout the immature brain, this phenomenon might be widespread in the developing central nervous system.

Neuronal Per Arnt Sim domain 4 (Npas4), a brain-specific helix-loop-helix transcription factor, was recently shown to regulate the development of GABAergic inhibitory neurons. Npas4 mRNA expression levels were decreased in the hippocampus of mice exposed to stress, which was accompanied by brain dysfunction. We have suggested that transient stress reduced Npas4 transcription through the glucocorticoid receptor. In the present report, we investigated the potential contribution of epigenetic modifications induced by stress on Npas4 gene expression. The Npas4 promoter region contains two CpG islands; in the hippocampus, chronic restraint stress increases the DNA methylation levels of both of these CpG islands. In the Neuro2a cell line, treatment with a DNA methyltransferase inhibitor, 5-aza-2-deoxycytidine, increased Npas4 mRNA levels and markedly reduced the DNA methylation levels of CpG island 2 in the Npas4 promoter. The DNA methylation sites in CpG island 2 overlap with two cyclic adenosine monophosphate response element (CRE) sequences. Mutation of these CRE sequences reduced Npas4 promoter activity. These results suggest that transcription of the Npas4 gene is downregulated by stress through DNA methylation of its promoter.

Reelin has recently attracted attention because of its connection to several neuropsychiatric diseases. We previously reported the finding that prior transplantation of GABAergic neuron precursor cells into the medial prefrontal cortex (mPFC) of mice significantly prevented the induction of cognitive and sensory-motor gating deficits induced by phencyclidine (PCP). The majority of the precursor cells transplanted into the mPFC of the recipient mice differentiated into members of a somatostatin/Reelin-expressing class of GABAergic interneurons. These findings raised the possibility that Reelin secreted by the transplanted cells plays an important role in preventing the deficits induced by PCP. In this study, we investigated whether Reelin itself has a preventive effect on PCP-induced behavioral phenotypes by injecting conditioned medium containing Reelin into the lateral ventricle of the brains of 6- to 7-week-old male mice before administrating PCP. Behavioral analyses showed that the prior Reelin injection had a preventive effect against induction of the cognitive and sensory-motor gating deficits associated with PCP. Moreover, one of the types of Reelin receptor was found to be expressed by neurons in the mPFC. The results of this study point to the Reelin signaling pathway as a candidate target for the pharmacologic treatment of neuropsychiatric diseases. (C) 2015 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

FUS is an RNA/DNA-binding protein involved in multiple steps of gene expression and is associated with amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar degeneration (FTLD). However, the specific disease-causing and/or modifying mechanism mediated by FUS is largely unknown. Here we evaluate intrinsic roles of FUS on synaptic functions and animal behaviours. We find that FUS depletion downregulates GluA1, a subunit of AMPA receptor. FUS binds GluA1 mRNA in the vicinity of the 3' terminus and controls poly (A) tail maintenance, thus regulating stability. GluA1 reduction upon FUS knockdown reduces miniature EPSC amplitude both in cultured neurons and in vivo. FUS knockdown in hippocampus attenuates dendritic spine maturation and causes behavioural aberrations including hyperactivity, disinhibition and social interaction defects, which are partly ameliorated by GluA1 reintroduction. These results highlight the pivotal role of FUS in regulating GluA1 mRNA stability, post-synaptic function and FTLD-like animal behaviours.