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3O2-1 Immune-related Factors Influenced Postnatal Synapse Remodeling in the Primate Cerebral Cortex Tetsuya Sasaki^1,2，Tomomi Sanagi^1,3，Tomoko Manabe¹，Shinichi Kohsaka⁴，Noritaka Ichinohe^1,2 ¹Dept Ultrastruc Res, Natl Inst Neurosci, NCNP，²Lab Molecular Analysis of Higher Brain Func, BSI, RIKEN，³RPD Fellowship of JSPS，⁴Natl Inst Neurosci, NCNP In primates, the number of dendritic spines rapidly increases after birth, reaches a peak at the early infancy, and then decreases towards the adult level. Abnormalities of the processes are implicated in several psychiatric disorders, such as ASD and schizophrenia. We investigated the normal processes of spine formation in the cerebral cortex of the common marmoset as a primate model. Our previous studies reported that all cortical areas examined showed overshoot-type spine formation with peaked at 2-3 month old (Oga et al., 2013, Sasaki et al., 2015). To clarify the molecular basis of spine pruning, we compared gene expression profile in area12, TE, and V1 at 3M (peak of spinogenesis) and 6M (pruning phase) using DNA microarray. Based on the criterion of at least 1.2 fold changes, we found that 1,577 genes showed differential expression between 3M and 6M. Canonical pathway analysis revealed that a large number of these genes belong to subsystems within the immune system. Among them, in particular, microglia-related genes were upregulated at 3M, and in contrast, genes belong to complement systems were upregulated at 6M. Immunohistological analysis indicated that density of ramified microglia with characteristic bulbous endings was increased at 3M. These results suggest that molecular and functional properties of microglia alter during spinogenesis/pruning period in primate cortex. To enable direct analysis of microglial properties ex vivo, we isolated microglia from marmoset brain using magnetic cell isolation method, which yielded a highly purified CD11b+cell population with properties that reflected their in vivo phenotype. Further research will enable the specific evaluation of microglia on primate-specific synapse remodeling process during postnatal period.		3O2-2 Microglial abnormality in the prefrontal cortex of a primate ASD model by prenatal exposure of valproic acid. Tomomi Sanagi^1,2，Tetsuya Sasaki^1,3，Tomoko Manabe¹，Keiko Nakagaki¹，Shinichi Kohsaka⁴，Noritaka Ichinohe^1,3 ¹Dept of Ultrastructural Res, Natl Inst of Neurosci, NCNP，²RPD Fellowship of JSPS，³Lab for Molecular Analysis of Higher Brain Function, RIKEN BSI，⁴Natl Inst of Neurosci, NCNP Microglia are supposed to be surveyors and scavengers in the brain. Recently, microglial abnormalities have been observed in the brains of patients with neuropsychiatric disorders including autism spectrum disorders (ASD), suggesting that microglia are implicated in the pathophysiology of these disorders. In the cerebral cortex of primates including human, the number of dendritic spines rapidly increases after birth, reaches a peak at around the infancy or mid-childhood, and decreases towards adulthood. In neuropsychiatric disorders, the abnormalities in the number of dendritic spines are one of the pathological findings. In ASD, excess spine formation and imperfect synaptic elimination (pruning) result in increased spine number. To reveal microglial involvements in the pathophysiology of ASD, we have developed ASD model of the common marmoset (Callithrix jacchus) by prenatal exposure to valproic acid (VPA). The VPA marmosets have a deficit in social interaction, which is one of features found in ASD patients (Yasue et al., 2015). In this study, we immunostained multiple cortical areas using anti-Iba1 antibody at 2M, 3M, 6M and adult, and investigated the developmental changes of microglia in VPA-exposed marmosets and unexposed (UE) marmosets. We found the morphological abnormality of microglia (e.g. decreased density, change in process form) in VPA marmosets compared with UE marmosets. Furthermore, we investigated the immunohistological changes of several genes related to microglial functions and inflammation. Our results support the microglial abnormalities observed in the brains of ASD patients. The VPA marmosets are useful primate model of ASD and further analysis using this model would lead to understand the microglial roles in the pathophysiology of ASD.

3O2-3 Evaluation of novel readthrough agents by using myelin P0-translatinal system in vivo Yoshinori Otani¹，Yoshihide Yamaguchi¹，Akihoro Taguchi²，Keisuke Hamada²，Yoshio Hayashi²，Hiroko Baba¹ ¹Department of Molecular Neurobiology, Tokyo University of Pharmacy and Life Sciences，²Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences Large myelin protein zero (L-MPZ) is an isoform of myelin protein zero (P0), containing additional 63 amino acids at the C-terminus, and produced by stop codon readthrough mechanism (Yamaguchi et al., 2012). Our previous study showed that the adhesion activity of L-MPZ is weaker than P0, suggesting that the ratio of P0 and L-MPZ in myelin is important for normal myelin structure. Recently, readthrough agents have been developed to suppress nonsense mutations in the genetic disorders. It is important to clarify the influence of readthrough agents on proteins naturally produced by this mechanism. Previously, we examined the activities of novel readthrough agents, negamycin analogues (Taguchi et al., 2014) by in vitro translation system, on P0 gene to choose the appropriate agent for in vivo experiments. In the examination using the cells with stable expression of human P0 mRNA, percent ratios of L-MPZ-positive cells were increased to 60% (~10% in control) by three negamycin derivatives. At present, our purpose is to clarify L-MPZ function using negamycin derivatives in vivo. In this study, the direct injection of TCP1109 (one of three negamycin derivatives) in sciatic nerves exhibited 1.3-fold increase of L-MPZ/P0 ratio compared to vehicle control by western blotting. The nerve conduction velocity and Beam walk test showed abnormality in a classical readthrough agent G418 treated group, but not in TCP1109 treated group. In addition, TCP1109 showed less damage to sciatic nerve than G418. However, significant decreasing of G-ratio was observed in TCP1109 treated nerve by Semi-thin morphological analysis. Thus, it is important to examine physiological influence of translational readthrough in the PNS myelin using these agents.		3O2-4 The somatic stemness of oligodendrocyte precursor cells requires thyroid hormone and hypoxia to activate Runx1 Yasuhito Tokumoto^1,2,3，Shinpei Tamaki⁴，Yasuaki Kabe^2,5，Keiyo Takubo^4,5，Makoto Suematsu² ¹Admission Center, Saitama Medical Univ，²Dept Biochem, Sch Med, Keio Univ，³JST-ERATO，⁴Dept Stem Cell Biol, NCGM，⁵AMED-CREST Adult mammalian central nervous system (CNS) contains a population of slowly dividing oligodendrocyte precursor cells (OPCs), known as adult OPCs. Adult OPCs constitute approximately 5% of the cells throughout the adult CNS, where they have a crucial role in remyelination following CNS damage throughout the life of animal, suggesting that adult OPCs are adult somatic stem cells in CNS. While, in rodent optic nerve, these adult OPCs develop from rapidly dividing perinatal OPCs after the second weeks of birth, the mechanisms underlying the perinatal-to-adult transition of OPCs remain unknown. Here we show that rapidly dividing perinatal rodent OPCs cultured with thyroid hormone (TH) or retinoic acid under hypoxia fall into quiescence and acquire adult OPCs-like characters. Cyclin-dependent kinase inhibitor (CDKI) p15/INK4b plays crucial roles for the TH-dependent deceleration of OPC’s cell cycle under hypoxia. The gene of transcription factor Klf9 serves as the direct target of TH-dependent signaling. Under hypoxic conditions, hypoxia-inducible factors (HIFs) mediates upregulation of the gene expression of runt-related transcription factor 1 (Runx1, also known as AML1), the master gene of hematopoietic stem cell development. Runx1 induces G1 arrest of OPCs through enhancing the TH-dependent p15/INK4b gene expression. As adult OPCs are the tissue specific somatic stem cells in CNS, the current results shed light on environmental requirements for quiescence of tissue specific somatic stem cells during their development from actively proliferating embryonic stem/progenitor cells.Scientific Reports 7, Article number: 1019 (2017) doi:10.1038/s41598-017-01023-9www.nature.com/articles/s41598-017-01023-9

3O2-5 Measurement of multiple radical scavenging activity as a diagnostic method for children with ASD Hideo Matsuzaki¹，Aki Hirayama² ¹Research Center for Child Mental Development, University of Fukui, Fukui, Japan，²Center for Integrative Medicine, Tsukuba College of Technology, Ibaraki, Japan Oxidative stress plays a central role in the pathogenesis of autism spectrum disorders (ASD). Though early treatments are important, ASDs are rarely diagnosed in children younger than 2 years, because diagnosis is based deeply on behavioral tests. We investigated the availability of the measurements of multiple free radical scavenging activity (MULTIS) as a non-behavioral diagnostic tool for ASD children. Serum samples from 23 ASD children and 34 neurotypical children (NT), age 3.9±0.8 y.o., were analyzed under informed consents. In MULTIS method, serum scavenging-activity profile against 5 reactive oxygen species, namely .OH, O2.-, RO., ROO. and 1O2 were measured using spin traps CYPMPO and TMPO (Oowada S et al., J Clin Biochem Nutr. 2012;51:117-21.). Scavenging activity against was significantly decreased in ASD group (approximately 55% of NT group mean value). Contrary to .OH, scavenging activities against O2.- and RO. were significantly increased in ASD group (approximately 120% and 157% of NT group mean values, respectively). No differences were observed in ROO. and 1O2 among the two groups. When diagnostic criteria for ASD was made using .OH, O2.- and RO. scavenging activities, the calculated sensitivity, specificity and positive likelihood ratio were 65%, 93% and 9.78, respectively. Moreover, when the data of ROO. and 1O2 were added, the positive likelihood ratio increased to 22.17. Measurements of serum multiple free radical scavenging activity by MULTIS method can be a very powerful non-behavioral tool for diagnosis of ASD children.		3O2-6 ER stress-dependent production of small peptides derived from ER stress sensors in neuronal cells. Koji Matsuhisa¹，Atsushi Saito¹，Yosuke Ohtake^1,2，Kanta Yanagida³，Masayasu Okochi³，Masaki Matsumoto^4,5，Keiichi Nakayama^4,5，Kazunori Imaizumi² ¹Dept Stress Prot Processing, Inst Biomed & Health Sci, Hiroshima Univ，²Dept Biochem, Inst Biomed & Health Sci, Hiroshima Univ，³Psy, Dept Integrated Med, Div Int Med, Osaka Univ Grad Sch Med，⁴Div Proteomics, Med Inst Bioreg, Kyushu Univ，⁵Dep Mol & Cell Biol, Med Inst Bioreg, Kyushu Univ Endoplasmic reticulum (ER) stress sensor BBF2H7 is a transmembrane transcription factor. It is cleaved at its transmembrane region in response to ER stress. The cleavage is regulated by site-1 and site-2 proteases (S1P and S2P), resulting in producing the N- and C-terminal fragments. We recently found that the small peptide fragments including partial membrane region between the N- and C-termini are generated under ER stress conditions. In this study, we decided the amino acid sequence and biological characters of the peptide. SK-N-SH cells stably expressing BBF2H7 were treated with 1μM of ER stressor thapsigargin and 10μM of proteasome inhibitor MG132 for 24h. The cell lysates were immunoprecipitated followed by western blotting using anti-BBF2H7-derived peptide antibody. Monomer (5.5kDa) and dimer (11kDa) bands of the peptide were gradually increased, suggesting that it is produced during ER stress and very aggregable. Transfectants expressing BBF2H7 mutated in S1P recognition site showed no production of the peptide. The peptide also failed to be detected in S2P-deficient CHO cells transfected with BBF2H7. To determine the amino acid sequence of the peptide, it was collected with immunoprecipitation, and subjected to peptide sequencing using Edman degradation method. The analysis revealed that the peptide is composed of 45 amino acids including 12 residues at the transmembrane domain of BBF2H7. In conclusion, we identified the small peptide fragment derived from ER stress sensor. The peptide is generated via the intramembrane proteolysis regulated by S1P and S2P under ER stress conditions, and has highly aggregative property. We are investigating the effects of the peptide on aggregation of Aβ  that may provide the link of the peptide with neurodegenerative diseases.