Poster Sessions

Poster Sessions グリア、ミエリン、神経免疫、その他

1P-34 Effects of the IVIg treatment on the mice lysolecithin-induced peripheral nerve demyelination Akiko Hayashi¹，Daigo Yanaoka¹，Takafumi Yamada¹，Yuka Nishibe¹，Naho Suzuki¹，Ayami Kawada¹，Hiroaki Takimoto²，Hiroko Baba¹ ¹Department of Molecular Neurobiology, Tokyo University of Pharmacy and Life Sciences，²Department of Biosciences, Kitasato University Intravenous IgG Kenketu glovenin(R)-I (IVIg) treatment is known to be effective on human immune-mediated neurophathies. IVIg treatment also ameliorates clinical score and conduction velocity in rat experimental autoimmune neurophathy (EAN). However the mechanisms of IVIg teratment in the peripheral nerve demyelination are still uncertain. To elucidate this mechanism, the effect of IVIg on lysolecithin-induced chemical demyelination was examined.ICR mice were intraneurally injected with 1% Lysorecithin into the sciatic nerves, and either IVIg or control saline was injected intravenously 24 hours later. Sciatic nerves were resected, and evaluations of demyelinating areas and infiltrating macrophages were performed. On the day 7 after lysolecithin injection, demyelinating areas of IVIg injected group were smaller than those of saline injected group. Numbers of CD68+Arginase1+ M2 macrophages were increased in IVIg injected group, while CD68+Arginase1- macrophages were increased in saline treated group. Depositions of human IgG were observed on myelin debris in the demyelinating lesions. By western blotting using IVIg, only a 36-kDa single band of large myelin protein zero (L-MPZ) was detected in rat sciatic nerve homogenates, suggesting that antibodies against L-MPZ in IVIg directly bound to the debris. These results indicate that IVIg treatment has protective effect on chemical demyelinating condition, and inducible effect of M2 macrophages in acute phase. Direct binding of IVIg to myelin debris may be involved in this modulation process.		1P-35 Disruption of paranodal axo-glial junctions causes accumulation of axonal IP₃R1 in Purkinje cells in CST-deficient mice Tomoko Ishibashi¹，Taiga Tsurifune¹，Saki Takahashi¹，Katsuhiko Mikoshiba²，Hiroko Baba¹ ¹Dept Molecular Neurobiology, Tokyo University of Pharmacy and Life Sciences，²Lab for Developmental Neurobiology, RIKEN, BSI To understand how paranodal axo-glial junctions (PNJs) regulate axonal functioning, we studied cerebroside sulfotransferase deficient mice (CSTko) that partially lack PNJs. CSTko show Purkinje axonal swellings after myelination, and IP₃R1-positive focal accumulations are the earliest finding therein. We investigated the localization of several proteins related to IP₃R1 in mitochondria-associated membranes (MAM) as well as in ER to better understand the significance of IP₃R1 accumulation in axonal swellings in PNJs disrupted axons.In MAM, IP₃R1 and VDAC interact via GRP75, and ER lipid raft-associated protein 2 (ERLIN2) is involved in IP₃R1 degradation. Immunohistochemistry with specific antibodies against these proteins showed positive staining in IP₃R1-positive swellings suggesting an alternation of the localization of molecules in MAM in CSTko Purkinje axons. However, not all IP₃R1-positive swellings were MAM-related-protein-positive confirming the accumulations of IP₃R1 as the earliest finding in these swellings. In 12-month aged CSTko we found a strong presence of an autophagosome marker LC3 in the IP₃R1-positive swellings and Purkinje cell (PC) bodies, and observed PC loss. We further investigated cerebella from CST^-/-/IP₃R1^+/- mice. There were clearly less axonal swellings in CST^-/-/IP₃R1^+/- mice compared to CST^-/-/IP₃R1^+/+ mice and the staining of LC3 was lower in the CST^-/-/IP₃R1^+/- mice swellings. The extent of PC loss was also less in the CST^-/-/IP₃R1^+/- mice. These findings indicate that the focal accumulation of IP₃R1 triggers Purkinje axonal swellings and PC loss in CSTko. Our results suggest that the formation of PNJs is a critical event for the maintenance of axonal calcium homeostasis by regulation of IP₃R1 localization in Purkinje axons.

1P-36 Olig2-lineage astrocytes comprises a subpopulation of astrocytes distinct from GFAP-positive ones in the adult mouse brain. Kouko Tatsumi，Ayami Isonishi，Shoko Morita-Takemura，Tatsuhide Tanaka，Akio Wanaka Dept Anat and Neurosci, Nara Med Univ Olig2, a basic helix-loop-helix (bHLH) transcription factor, persists in the central nervous system from embryonic to adult stages. In the last decade, lineage tracing studies of Olig2-positive cells revealed that they generate mainly OPCs/NG2 glia, mature oligodendrocytes and a small number of astrocytes, but no neurons, in the adult brain under physiological condition. We have demonstrated that Olig2-lineage astrocyte (Olig2-AS) that were detected by GFP immunohistochemistry in Olig2 ^CreER: ROSA-GAP43-EGFP mice changed their morphology in response to overall running activities in the adult globus pallidus (Tatsumi et al. Front. Cell. Neurosci. 2016).In the present study, we mapped whole brain distribution of the Olig2-AS. Olig2-AS exhibited bushy morphology like protoplasmic astrocytes and co-expressed some mature astrocytic markers such as s100beta, 3-phosphoglycerate dehydrogenase (3-PGDH), and Glutamine synthetase (GS), but hardly co-expressed GFAP, the most popular astrocytic marker. Interestingly, Olig2-AS were not distributed evenly, but rather clustered in specific brain nuclei such as the nucleus of diagonal band, subthalamic nucleus, hypothalamic nuclei, thalamic pretectal nucleus, deep cerebellar nuclei and brain stem motor nuclei. This distribution pattern was distinct from that (GFAP-AS) detected in GFAP^cre: ROSA-GAP43-EGFP mice. Even in a single nucleus such as the globus pallidus and substantia nigra reticulata, Olig2-AS and GFAP-AS showed mutually-exclusive distribution patterns. Olig2-AS thus comprises a subpopulation of astrocytes distinct from GFAP-positive ones in the adult brain.		1P-37 Effect of functional fatty acids on proliferation of the mouse cultured astrocytes. Hitoshi Inada，Inês dos Reis，Noriko Osumi Dept Develop Neurosci, Grad Sch Med, Tohoku Univ Fatty acids are not only major components of lipid membrane or energy source for ATP production in mitochondria but also signaling molecules in various metabolism. Arachidonic acid (ARA), for example, is cleaved from membrane lipids and metabolized into lipid mediators, eicosanoids such as prostaglandins and thromboxanes, in the various biological responses including inflammation, allergy, vasoconstriction, and other immune responses. Recently, some functional fatty acids, especially essential polyunsaturated fatty acids (PUFAs) have been reported to play important roles in brain development and function. Arachidonic acid administration enhanced neurogenesis in hippocampus of juvenile rats and improved impaired prepulse inhibition, an endophenotype of psychiatric diseases, in adult rats. In this study, we focused on the effect of fatty acids on proliferation and function of astrocytes, i.e., the most abundant cells in the brain. Influence of three major PUFAs, arachidonic acid, docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) were investigated in regard with proliferation of cultured murine astrocytes. DHA supplement to the culture medium resulted in a tendency of increased proliferation, while AA or EPA supplement showed no significant effect. To further identify novel functional fatty acids affecting astrocyte proliferation and function, we screened 80 fatty acids with diverse biological activities. As a result, we identified twenty candidates with positive effect and four candidates with negative effect on astrocyte proliferation. These finding may imply significant roles of PUFAs in astrocytic functions.

1P-38 Phylogenetically conserved sequence around PNS myelin P0 stop codon is essential for the synthesis of the readthrough isoform L-MPZ Yoshihide Yamaguchi，Yu Naito，Yukiko Uchino，Mai Mogi，Hiroko Baba Dept Mol Neurobiol, Sch Pharm, Tokyo Univ Pharm & Life Sci Myelin protein zero (P0, MPZ) is phylogenetically highly conserved protein (amino acid homology to human P0: >90% in mammals, >70% in chicken, >60% in frog, >45% in fishes) expressed in Schwann cells in the PNS and involved in the myelin formation. Previously, we reported that large myelin protein zero (L-MPZ) is a stop codon readthrough isoform of P0, containing an additional domain at the C terminus. Surprisingly, the amino acid sequences of L-MPZ-specific region after canonical P0 stop codon are limitedly conserved from frog to human but not in fish. In evolution, P0 is distributed in both the PNS and the CNS in fish, but only in the PNS after amphibian. Since the sequences around canonical stop codon in P0 mRNA are also highly conserved after amphibians in the evolutional progression, these sequences expected to be related to stop codon readthrough. The relationship between sequence around stop codon and L-MPZ production is as yet unclear. While we have demonstrated the production of L-MPZ in rats and mice, actual production of L-MPZ has not been assed in frog and human. In this study, we demonstrated the presence of L-MPZ in frog and human by Western blotting using L-MPZ-specific antibody. The mutant analysis using mammalian in vitro transcription/translation system revealed that the evolutionally conserved 3 nucleotide (AAA) just before P0 stop codon was more susceptible to readthrough. Additionally, even the frog mRNA–specific type containing 3 nucleotide (CAA) just after the P0 stop codon exhibited almost the same or higher readthrough activity than the conserved CGG type in other species. Thus, the evolutionarily conserved context of sequence around stop codon is essential for the specific synthesis of L-MPZ by translational readthrough after amphibian.		1P-39 The absence of myelin basic protein reduces the non-amyloidogenic APP processing pathway in the MBP^-/- mice and bigenic Tg2576/MBP^-/- mice Chika Seiwa^1,2，Saki Nakashima³，Hiroaki Asou^2,3 ¹Lab of Mol Neurosci and Neurol Sch of Life Sci, Tokyo Univ of Pharmacy and Life Sci，²Glovia Myelin Res Inst，³Keio univ Sch of Med Amyloid- β peptide (Aβ) has been suggested to be causal in Alzheimer’s disease (AD). Aβ monomers assemble into oligomers and fibrils that promote neuronal dysfunction. Amyloid precursor protein (APP) is cleaved by protease called α-, β-, and γ-secretase.  The subsequential cleavages by β- and γ- secretase generate the amyloidogenic Aβ peptides and simultaneously soluble β-APP (sAPPβ).  In contrast, α-secretase cleavage precludes Aβ formation and generate soluble α-APP (sAPPα) . A recent study demonstrates that myelin basic protein (MBP) has been shown to inhibit amyloid fibril formation via its direct binding to and degradation of amyloid by an intrinsic protease activity. In this study, we investigated whether the absence of MBP could influence of APP processing pathway. We took advantage of MBP^-/- mice, known as shiverer mice, in which are unable to synthesize any of the MBP isoforms causing severer CNS hypomyelination and by breeding shiverer mice with Tg2576, mouse model of AD. We found that shiverer MBP^-/-  mice and bigenic Tg2576/ MBP^-/- mice had a significant increased of hexameric soluble Aβ oligomer compared to MBP normal control wild type mice. In addition, there was a significant reduction in cerebral sAPPα levels of the α-secretase ADAM9, catalyses non-amyloidogenic processing of the APP, since MBP strongly binds ADAM 9 and enhanced amount or activity of α-secretase in the normal brain. Thus, there results have suggested that the absence of MBP decrease non-amyloidgenic APP processing sAPP α and targeting oligodendrocytes / myelin may be a novel therapeutic intervention for the prevention and treatment of AD.

1P-40 Loss of p73 in ependymal cells during the perinatal period leads to aqueductal stenosis Masashi Fujitani^1,2，Toshihide Yamashita²，Koichi Noguchi¹ ¹Department of Anatomy and Neuroscience, Hyogo College of Medicine，²Dept Molecular Neuroscience, Grad Sch Med, Osaka Univ The p53 family member p73 plays a critical role in brain development. p73 knockout mice exhibit a number of deficits in the nervous system, such as neuronal death, hydrocephalus, hippocampal dysgenesis, and pheromonal defects. Among these phenotypes, the mechanisms of hydrocephalus remain unknown. Here, we generated a p73 knock-in (KI) mutant mouse and conditional p73 knockout mouse. The homozygous KI mutants showed aqueductal stenosis, which is not observed in conventional knockout mice. p73 was expressed in the ependymal cell layer and several brain areas. An assessment of the integrity of the cilia and basal body (BB) patch formation suggests that p73 is required to establish translational polarity but not to establish rotational polarity or the planar polarization of BB patches. However, deletion of p73 in adult ependymal cells did not affect the maintenance of translational polarity. Importantly, when p73 was disrupted during the perinatal period, animals showed aqueductal stenosis at a later stage. These results suggest that the loss of p73 during the perinatal period is critical for hydrocephalus development.		1P-41 TRPM2 channel aggravates the CNS inflammation via increased production of CXCL2 chemokines in experimental autoimmune encephalomylitis Hisashi Shirakawa，Ryo Hirase，Masato Tsutsui，Sakie Miyamura，Kazuki Nagayasu，Shuji Kaneko Dept Mol Pharmacol, Grad Sch Pharm Sci, Kyoto Univ. Multiple sclerosis (MS) is a chronic inflammatory disorder of the CNS characterized by demyelination and axonal injury. Current therapies for MS mainly targeting whole lymphocytes do not fully meet the clinical needs because of the severe side effect risks and the insufficient effect on the progressive MS. Accumulating evidence suggests that CNS inflammation is associated with this demyelinating disease, but the molecular identity responsible for their activation is poorly understood. Transient receptor potential melastatin 2 (TRPM2) is a Ca²⁺-permeable nonselective cation channel that is expressed highly in the brain and immune cells. Here we show the involvement of TRPM2 in the pathology of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. We found the gene deletion or the pharmacological inhibition of TRPM2 inhibited the progression of EAE and the activation of Iba1-immunopositive monocyte lineage cells and the infiltration of neutrophils were suppressed in the CNS of TRPM2-KO mice compared with wild-type (WT) mice. Moreover, we demonstrate that the preceding reduction of CXCL2 in the CNS of TRPM2-KO mice at the time point when the EAE severities were not different between WT and TRPM2-KO mice, and that TRPM2 expressed in CNS-infiltrated macrophages contributed to the progression of EAE by using radiation bone marrow chimeric mice. Since CXCL2 is well known as a chemokine inducing the migration of neutrophils, those results indicate that the reduction of CXCL2 in the CNS caused the suppressed infiltration of neutrophils and the restrained the progression of EAE in TRPM2-KO mice. Taken together, here we report the important roles of TRPM2 in the progression of EAE pathology and shed light on TRPM2 as the therapeutic target of MS.

1P-42 Microglia support ATF3-positive neurons following hypoglossal nerve axotomy Tatsuhide Tanaka¹，Koichi Murakami²，Yoshio Bando²，Taichi Nomura²，Ayami Isonishi¹，Shoko Morita-Takemura¹，Kouko Tatsumi¹，Akio Wanaka¹，Shigetaka Yoshida² ¹Dept Anat. and Neurosci., Nara Med. Univ.，²Dept Functional Anat. and Neurosci., Asahikawa Med. Univ. Microglia are essential in developmental processes and maintenance of neuronal homeostasis. Experimental axotomy of motor neurons results in neurodegeneration, and microglia in motor nuclei become activated and migrate towards injured neurons. However, whether these activated microglia are protective or destructive to neurons remains controversial. In the present study, we transected the hypoglossal nerve in BALB/c mice, causing activating transcription factor 3 (ATF3) and growth associated protein 43 (GAP43) induction, and partial neuronal death. Inhibition of microglial accumulation by minocycline administration impaired microglial accumulation, decreased GAP43 mRNA expression, and reduced motor neuron survival. Expression of ATF3 contributed to nerve regeneration, and increased within 6 hours after axotomy, prior to microglial migration. Further, microglial contact with neuronal cell bodies was associated with neuronal ATF3 expression. Colchicine administration blocked lesion-induced ATF3 transcription in axotomized neurons and microglial accumulation. In addition, perineuronal microglia-derived ciliary neurotrophic factor (CNTF) increased, indicating that perineuronal microglia in the hypoglossal nucleus protect axotomized motor neurons by releasing trophic factors. We also observed that microglia secrete CNTF and that neurons have CNTFRα and can respond to it in vitro. CNTF promote neurite elongation and neuronal survival of primary cultured neurons. Microglia make contact through unknown neuronal signals that are possibly regulated by ATF3 in hypoglossal nucleus. Moreover, they play important roles in regenerating motor neurons and are potential new therapeutic targets for motor neuron diseases.		1P-43 DHA-dependent lipid peroxidation increases α-synuclein aggregation and accumulation via inhibition of autophagy system in neuronal cells Masayo Shamoto-Nagai，Wakako Maruyama，Makoto Naoi Dept Health Nutr. Fac Psychol & Physical Sci. Aichi Gakuin Univ Accumulation of modified α-synuclein (αSyn) is one of the pathogenic factors for Parkinson disease (PD). The autophagy-lysosome system is the major site to degrade damaged αSyn, and might be associated with PD. Compartment formation of targeted protein with membrane initiate the proteolysis in the system and age-dependent oxidative stress increases the oxidation and modification of membrane lipid should impair the function and induce protein accumulation in neurodegenerative disorders. This paper presents the effects of lipid peroxidation on the autophagy-lysosomal system in relation to αSyn accumulation in the brain. Lipid peroxidation was induced by docosahexaenoic acid (DHA), a major polyunsaturated fatty acid in the brain, in human neuroblastoma SH-SY5Y cells transiently expressing αSyn (SYN-SH cells). The activity of autophagosome and lysosome was determined by reduction of specified fluorescent markers. Overexpression of αSyn inhibited lysosomal formation. In addition, DHA (20μM) treatment inhibited the lysosome and formation of autophagosome in SYN-SH cells. αSyn was markedly accumulated and co-localized with LC3B, a specific marker for the autophagosome. On the other hand, neither  αSyn overexpression nor DHA alone affected the lysosome-autophagosome fusion. These results indicate that lipid-peroxidation induced by exogenous or excess DHA may perturb proteolysis of αSyn in the autophagy-lysosome system. The role of lipid peroxidation is discussed in relationship to pathogenic protein accumulation in the neurons in ageing and neurodegenerative disorders.

1P-44 Differential changes of drebrin and PSD-95 localization in response to glutamate stimulation Noriko Koganezawa，Yukai Chiu，Tomoaki Shirao DNBB, Grad Sch Med, Gunma Univ Dendritic spines are postsynaptic responsive regions of excitatory synapses and play an important role in synaptic transmission and plasticity. Several proteins which localize within dendritic spines are known to regulate spine morphology and function, and the subcellular distribution changes of these proteins are thought to be important factors of synaptic plasticity. To reveal nanoscale organization of synaptic proteins, we used stochastic optical reconstruction microscopy (STORM). We first observed localizations of several synaptic proteins such as drebrin, PSD-95 and synapsin I during resting state using primary hippocampal cultured neurons. Both drebrin and PSD-95 exist at postsynaptic site and synapsin I presents at presynaptic site. In the STORM images, drebrin accumulated in the center of the spine heads and synapsin I localized face to face. PSD-95 also localized within the dendritic spines, but it has slightly different distribution pattern from drebrin. It is known that NMDA receptor activation induces a bidirectional shift in subcellular distribution of drebrin. Thus we further observed localization changes of these proteins after glutamate stimulation. As a result, drebrin changed its distribution from dendritic spine heads to the dendritic shafts after the glutamate stimulation, while PSD-95 stayed in the spine heads. Synapsin I did not change its distribution pattern similar to PSD-95. These results suggest the presence of a stable core of F-actin, which is formed by drebrin, in the central region of the spine head. Drebrin exodus from spine heads after glutamate stimulation indicated loss of stable F-actin and dynamic F-actin is thought to be predominant. Dynamic localization changes of drebrin indicate an important role of drebrin in spine plasticity.		1P-45 Sweetness-induced rat brain activity changes and comparative examination of sucrose and artificial sweetener Yukiko Kondo¹，Satomi Higuchi²，Ikuko Uwano²，Fumio Yamashita²，Makoto Sasaki²，Masamichi Hirose³，Eiichi Taira¹ ¹Dept Pharmacol. Sch Med, Iwate Med Univ，²Div Ultrahigh field MRI. Inst Biomed Sci, Iwate Med Univ，³Dept Mol Cell Pharmacol. Sch Pharm Sci, Iwate Med Univ Artificial sweeteners have been used for the aim of diminishing the calories of the foods and beverages, instead of sugar. The artificial sweetener has strong sweetness even in small quantities and is low-calorie. Artificial sweeteners are used for suppression of weight gain in patients with obesity and diabetes. However, from the results of animal experiments, excess intake of artificial sweeteners suggests a cause of overeating, weight gain, increase in body fat percentage and increase in diabetes risk. In addition, even the results of human studies, excessive intake of artificial sweeteners cause weight gain, increase in visceral fat and obesity increase, and has been reported to increase in high blood pressure, diabetes, metabolic syndrome and depression as a result. However, it is not clear whether artificial sweetener intake really causes these events. Therefore, we examined the time course of weight gain of mice fed by foods with sweetened water. We also examined brain activity of rats by functional magnetic resonance imaging (fMRI) when rat tongue was stimulated with sweeteners. We used acesulfame K and sucrose as a sweetener. The acesulfame K is artificial sweetener with 200 times as much as sweetness as sugar and is a zero calorie. It is also one of the artificial sweeteners that are used in many foods and beverages currently. As a result, the mice were found to prefer acesulfame K or sucrose than water. In addition, it was suggested that the rats like also acesulfame K as much as sucrose, up to a certain fixed concentration. There was no little difference in weight gain between mice that ingested sucrose or acesulfame K. However, from the result of fMRI in rats, it was shown that cerebral activation area changed considerably with each sweetener stimuli.