神経化学教育口演

神経化学教育口演 Glia・Myelin・Neuronal Death・Apoptosis

1G2-01 Analysis of axon selective myelination depending on neuronal subtypes and neuronal activity Osanai Yasuyuki^1,2，Shimizu Takeshi^1,2，Mori Takuma^2,3，Yoshimura Yumiko^2,3，Hatanaka Nobuhiko^2,4，Nambu Atsushi^2,4，Kobayashi Kenta^2,5，Ikenaka Kazuhiro^1,2 ¹Division of Neurobiology and Bioinformatics, National Institute for Physiological Sciences（NIPS），²Department of Physiological Sciences, School of Life Sciences, Graduate University for Advanced Studies，³Division of Visual Information Processing, NIPS，⁴Division of System Neurophysiology, NIPS，⁵Section of Viral Vector Development, NIPS Oligodendrocyte（OL）myelinates multiple axons in the central nervous system. Recent reports have revealed that depolarization of myelinating OL increases conduction velocity of axons. OLs are also known to transfer exosomes to neuronal axons that respond to neurotransmitters. These findings suggest OLs modulate functions of multiple neurons, because OLs ensheath a lot of axons. If one OL selectively ensheaths particular neuronal axons, it is possible that the OL comprehensively modulate functions of those neurons. We examined whether OL preferentially myelinate a particular type of axons depending on neuronal subtypes or neuronal activity. We previously developed a novel method to observe interaction between each OL and neuronal axons. In this method, injection of rabies virus harboring the gene encoding GFP sparsely labels OLs in the targeted white matter, adeno-associated virus type2 labels axons projecting to the white matter massively. By the method, we found 25.9% of callosal OLs preferentially ensheath axons derived from particular brain areas and 50% of chiasmal OLs dominantly ensheath axons derived from either of two eyes. In addition, unilateral eyelid suturing does not impact on myelination by each chiasmal OL. This study revealed that some parts of OLs selectively myelinate axons depending on neuronal subtypes, and suggesting OLs do not select axons depending on neuronal activity.		1G2-02 Bergmann glia alignment along the Purkinje cell layer is important for the proper translocation of climbing fiber synapses from the Purkinje cell soma to dendrites Kikuchihara Saori^1,2，Inamura Naoko^1,5，Sugio Shouta^1,6，Tanaka Kenji³，Watanabe Masahiko⁴，Ikenaka Kazuhiro^1,2 ¹Div. of Neurobiology and Bioinformatics, National Institute for Physiological Sciences，²Dept. of Physiological Sciences, The Graduate University for Advanced Studies, School of Life Science，³Dept. of Neuropsychiatry, Keio University School of Medicine，⁴Dept. of Anatomy and Embryology, Hokkaido University School of Medicine，⁵Div. of Neuropathology, Department of Pathology, Institute for Developmental Research, Aichi Human Service Center，⁶Dept. of Molecular and Cellular Neurobiology, Gunma University Graduate School of Medicine The climbing fiber（CF）is a specific type of projection providing a powerful input to the cerebellum from the inferior olive. CF to Purkinje cell（PC）synapses have been well studied as a model for the process of synapse elimination in the central nervous system. Although increasing reports suggest that astrocytes contribute on synapse pruning in other brain regions, role of Bergmann glia（BG）, a type of astrocyte specific to the cerebellum, in CF synapse elimination is poorly understood. BG align their soma in the Purkinje cell layer（PCL）and have a fiber attaching with pia matter via their endfeet. In our previous study, transgenic mice overexpressing an astrocyte specific membrane protein MLC1 in astrocyte lineage cells showed abnormal alignment of BG in the molecular layer. MLC1 is one of the causative genes for leukoencephalopathy. Further analysis on the effect of MLC1 overexpression in cerebellar development revealed that MLC1 overexpression disturbed BG alignment only after birth. In mature BG, MLC1 expression was partially associated with Purkinje cell soma. Interestingly, a marker for climbing fiber synapse（vGluT2）and MLC1 had their own distinct territories in PCL. In the MLC1 overexpressing mice, MLC1 appeared to tightly wrap the Purkinje cell soma. Moreover, elimination of CF synapses to their dendrites was impaired. Though PC dendrites existed, translocation of CF synapses to them was seemed to be inhibited. These results suggest that BG alignment along the PCL is important for the proper translocation of CF synapses from the PC soma to dendrites.

1G2-03 In vivo Ca²⁺ imaging reveals that spinal astrocytes respond to nociceptor stimulation Matsuda Tsuyoshi¹，Tozaki-Saitoh Hidetoshi¹，Mikoshiba Katsuhiko²，Inoue Kazuhide³，Tsuda Makoto¹ ¹Dept. Life Innovation, Grad. Sch. Pharmaceut. Sci, Kyushu Univ.，²Lab. For Developmental Neurobiology, RIKEN Brain Science Institute，³Dept. Mol. Syst. Pharmacol., Grad. Sch. Pharmaceut. Sci, Kyushu Univ. Astrocytes, a major type of glial cells in the CNS, respond to neuronal activity, elevate intracellular Ca²⁺ levels and modulate synaptic transmission by astrocytic factors released in a Ca²⁺-dependent manner. The spinal dorsal horn（SDH）directly receives sensory inputs from the periphery through primary afferent fibres, but there is no report showing that astrocytes in the SDH respond to sensory inputs through primary afferents in vivo. In this study, we utilized an in vivo Ca²⁺ imaging technique and monitored activity of mouse SDH astrocytes that had expressed the ultrasensitive calcium sensor protein GCaMP6 by microinjecting with an AAV encoding this protein. We found that intracellular Ca²⁺ levels in SDH astrocytes were clearly increased soon after an intraplantar injection of capsaicin, a TRPV1 agonist that stimulates nociceptors. SDH astrocytes also responded to noxious mechanical stimulation（pinch）, but not innocuous stimuli（touch and acetone）. The capsaicin and pinch-induced astrocytic Ca²⁺ elevations were diminished by ablation of TRPV1-positive primary afferent fibers using resiniferatoxin, indicating that TRPV1-positive fibers transfer noxious information to SDH astrocytes. Furthermore, we also found that the noxious stimulation-evoked astrocytic Ca²⁺ signals disappeared in mice lacking inositol 1,4,5-trisphosphate（IP₃）receptor type 2（IP₃R2）. Taken together, SDH astrocytes are activated following noxious stimulation in a manner that requires IP₃R2 and may contribute to pain processing in the SDH.		1G2-04 Cereblon accumulates in aggresome and shows cytoprotective effect against proteasome inhibition Matsumoto Kodai¹，Wakabayashi Satoru¹，Yamada Haruka¹，Asahi Toru^1,2，Sawamura Naoya^1,2 ¹Faculty of Science and Engineering, Waseda University，²Research organization for nano-life innovation A nonsense mutation of the gene cereblon was found in a large kindred associated with intellectual disability（Higgins J.J. et al. Neurology 63, 1927-1931（2004））. The gene product cereblon（CRBN）is reported as a component of E3 ubiquitin ligase complex（Ito T et al. Science 327, 1345-1350（2010））. E3 ubiquitin ligase is known as a key factor to form aggresome. Aggresome formation is one of the defense mechanisms in living cells under various stress conditions（Olzmann J.A. et al. Biochem. So. Trans. 38, 144-149（2010））. Furthermore, it is reported that the aggregated protein is degraded by autophagy after aggresome formation at the perinuclear region. Here we examined whether CRBN accumulates in aggresome under proteasome inhibition. CRBN overexpressing PC12 cells were treated with proteasome inhibitor MG132. Exogenous CRBN localized at perinuclear regions, and co-localized with several aggresome markers under MG132 treatment. Moreover, we examined whether CRBN overexpressing PC12 cells shows cytoprotective effects against protease inhibition. Cell death induced by MG132 were significantly decreased in CRBN overexpressing PC12 cells compared with that of control cells. We propose that CRBN accumulates in aggresome and shows cytoprotective effects against protease inhibition.

1G2-05 Mitochondrial-targeted cereblon suppressed stress-induced cell death Kataoka Kosuke¹，Asahi Toru^1,2，Sawamura Naoya^1,2 ¹Faculty of Science and Engineering, Waseda University，²Research organization for nano-life innovation Cereblon is a candidate gene for autosomal recessive form of mild mental retardation（Higgins JJ et al., Neurology 63, 1927-1931（2004））. The product Cereblon（CRBN）contains a conserved Lon domain, which is a characteristic domain found in Lon protease（Higgins JJ et al., Neurology 63, 1927-1931（2004））. Lon protease is one of the stress response proteins in mitochondria in mammalian cells（Venkatesh S. et al., Biochimica et Biophysica Acta-Molecular Cell Research 1823, 56-66（2012）, Jenny K. Ngo et al., Free Radical Biology and Medicine 46, 1042-1048（2004））. Under various extracellular stresses, such as oxidative stress and nutrient starvation, Lon protease is up-regulated and supports the cell viability（Jenny K. Ngo et al., Free Radical Biology and Medicine 46, 1042-1048（2004））. Although CRBN contains a highly conserved, large Lon domain, whether CRBN has Lon protease-like functions remains unknown. Here, we investigated whether CRBN, similar to Lon protease, plays a protective role against extracellular stresses. Firstly, using western blot and confocal microscopy analysis, we reported that CRBN was present in various subcellular compartments including mitochondria. Next, to focus on the mitochondria-specific function of CRBN, we constructed an expression vector of mitochondria targeting sequence（MTS）-fused CRBN. Using this vector, we constructed stable human neuroblastroma SH-SY5Y cell lines expressing MTS-CRBN. Finally, we confirmed that the cell lines showed suppression of neuronal cell death induced by hydrogen peroxide and serum starvation. Taken together, these results indicate that mitochondrial-targeted CRBN could be responsible for the protective functions against extracellular stresses, as Lon protease does.		1G2-06 Nuclear cereblon modulates Ikaros-mediated transcription Wada Takeyoshi¹，Asahi Toru^1,2，Sawamura Naoya^1,2 ¹Faculty of Science and Engineering, Waseda University，²Research organization for nano-life innovation, Waseda University The gene coding for cereblon（CRBN）was originally identified in genetic linkage analysis for mild autosomal recessive nonsyndromic mental retardation. CRBN was also identified as a thalidomide-binding protein and a component of cullin-4-contaning E3 ubiquitin ligase complex（Ito et al., Science 327, 1345-50（2010））. CRBN has broad localization in both the cytoplasm and the nucleus. However, the significance of nuclear CRBN remains unknown. In the present study, we aimed to elucidate the role of CRBN in the nucleus. Firstly we generated a series of CRBN deletion mutants and determined the regions responsible for the observed nuclear localization. Only CRBN protein lacking the N-terminal region（1-119 a.a.）was localized outside the nucleus, suggesting that the N-terminal region is important for its nuclear localization. Thalidomide has been reported to be involved in the regulation of a transcription factor, Ikaros, with CRBN mediated degradation（Lu et al., Science 343, 305-9（2014）；Kronke et al., Science 343, 301-5（2014））. To further investigate the nuclear function of CRBN, we performed co-immunoprecipitation experiments and evaluated the association between CRBN and Ikaros. We showed that CRBN is associated with Ikaros protein, and the N-terminal region of CRBN was required for Ikaros binding. Using luciferase reporter gene experiments, we showed that CRBN modulates transcriptional activity of ikaros. These results suggested that CRBN regulates transcriptional activity of Ikaros, and may play an important role in the neuronal development involved in this transcription factor.