一般演題（口述）

一般演題（口述） Neuronal Degeneration

1O2-01 Rer1 and calnexin regulate endoplasmic reticulum retention of a peripheral myelin protein 22 mutant that causes type 1A Charcot-Marie-Tooth disease Hara Taichi，Hashimoto Yukiko，Akuzawa Tomoko，Hirai Rika，Kobayashi Hisae，Sato Ken Laboratory of Molecular Traffic, Institute for Molecular and Cellular Regulation, Gunma University Charcot-Marie-Tooth disease（CMT1）is the most commonly inherited neurological disorder of the peripheral nervous system with an estimated frequency of 1/2,500. Approximately 70% of patients with CMT1 harbor a genetic abnormality（e.g. duplication and mutation）of a membrane protein, PMP22. Although the accumulation of misfolded PMP22 in the endoplasmic reticulum（ER）correlates with pathogenic mechanism, the molecular mechanisms in the ER accumulation of PMP22 are largely unknown. Here, we studied the quality control mechanisms for the PMP22 mutants L16P and G150D, which were originally identified in mice and patients with CMT. We found that the ER-localised ubiquitin ligase Hrd1/SYVN1 mediates ER-associated degradation（ERAD）of PMP22（L16P）and PMP22（G150D）, and another ubiquitin ligase, gp78/AMFR, mediates ERAD of PMP22（G150D）as well. We also found that PMP22（L16P）, but not PMP22（G150D）, is partly released from the ER by loss of Rer1, which is a Golgi-localised sorting receptor for ER retrieval. Rer1 interacts with the wild-type and mutant forms of PMP22. Interestingly, release of PMP22（L16P）from the ER was more prominent with simultaneous knockdown of Rer1 and the ER-localised chaperone calnexin than with the knockdown of each gene. These results suggest that CMT disease-related PMP22（L16P）is trapped in the ER by calnexin-dependent ER retention and Rer1-mediated early Golgi retrieval systems and partly degraded by the Hrd1-mediated ERAD system.		1O2-02 Multivesicular body is formed after endoplasmic reticulum stress. Kanemoto Soshi，Matsuhisa Koji，Cui Min，Imaizumi Kazunori Department of Biochemistry, Institute of Biomedical and Health Sciences, Hiroshima University A number of cellular stress conditions lead to the accumulation of unfolded or misfolded proteins in the endoplasmic reticulum（ER）lumen, named as ER stress. Prolonged ER stress results in a fundamental threat to the cell. Accumulation of malfolded proteins in the ER triggers the unfolded protein response（UPR）to avoid cell damages. The UPR consists of at least three distinct components, namely transcriptional induction of ER-resident chaperones, translational attenuation, and ER-associated degradation（ERAD）. A number of studies indicate that ER stress and its stress response are associated with pathophysiology of neurodegenerative disorders including Alzheimer’s disease and Parkinson’s disease. In this study, we found that multivesicular body（MVB）was formed in response to ER stress. MVBs are a type of late endosome containing intraluminal small vesicles which include secretary proteins, and the vesicles in MVBs are secreted as exosomes. Treatment of human neuroblastoma SK-N-SH cells and human glioma U251MG cells with ER stressors, such as thapsigargin and tunicamycin, enhanced formation of MVBs that are detected by a MVBs marker, GFP-TSG101. We also investigated whether three major ER stress transducers, IRE1, PERK, and ATF6 are involved in the formation of MVBs. In PERK knockout mouse embryonic fibroblasts（MEFs）, MVBs were not formed by the treatment with thapsigargin, whereas MVBs were formed in IRE1αβ knockout or ATF6αβ knockdown MEFs after treatment with thapsigargin. Activation of eIF2α downstream of PERK pathway by a chemical compound salubrinal also increased MVB formation, suggesting ER stress facilitates the formation of MVBs through the activation of PERK-eIF2α pathway. Inhibition of MVB formation by the treatment with manumycinA which is a neutral sphingomyelinase inhibitor induced the expression of UPR related genes such as BiP, EDEM, and CHOP. Our findings suggest that MVB formation in response to ER stress is regulated via PERK-eIF2α signaling and might function to attenuate ER stress.

1O2-03 Sigma 1 receptor deficiency is involved in motor neuronal degeneration through calcium deregulation at mitochondria-associated membrane. Watanabe Seiji，Yamanaka Koji Research Institute of Environmental Medicine, Nagoya University A homozygous mutation in the gene coding sigma 1 receptor（Sig1R）is causative for juvenile inherited amyotrophic lateral sclerosis（ALS）. Sig1R specifically localizes at an interface of mitochondria and endoplasmic reticulum called as mitochondria-associated membrane（MAM）. In this study, we aimed to elucidate the mechanism that a loss-of-function of Sig1R causes ALS. First, ALS-linked Sig1R mutant was unstable and unable to bind to inositol triphosphate receptor type 3（IP₃R3）. Loss of Sig1R resulted in mislocalization of IP₃R3 and deregulation of intracellular calcium flux. In ALS-linked mutant Cu/Zn superoxide dismutase（SOD1）transgenic mice, mutant SOD1 proteins were accumulated in MAM, inducing depletion of Sig1R and IP₃R3 from MAM. Moreover, onset of the disease for SOD1^G85R mice was markedly accelerated in the absence of Sig1R with over-activation of calpain. Our findings suggest that the loss-of-interaction between Sig1R and IP₃R3 causes motor neuronal degradation through calcium deregulation in MAM, and restoring the function may be a promising therapeutic strategy.		1O2-04 Dextran sulfate sodium inhibits amyloid-β oligomer binding to cellular prion protein Aimi Takahiro¹，Hoshino Tatsuya²，Mizushima Tohru¹ ¹Department of Drug Discovery and Development, Faculty of Pharmacy, Keio University，²International University of Health and Welfare Amyloid-β peptide（Aβ）, especially its oligomeric form, is believed to play an important role in the pathogenesis of Alzheimer’s disease（AD）and the binding of Aβ oligomer to cellular prion protein（PrP^C）plays an important role in synaptic dysfunction in a mouse model of AD. In this study, we have screened for compounds that inhibit Aβ oligomer binding to PrP^C from medicines already used clinically, and identified dextran sulfate sodium（DSS）. In a cell-free assay, DSS inhibited Aβ oligomer binding to PrP^C but not to ephrin receptor B2, another endogenous receptor for Aβ oligomers, suggesting that the drug’s action is specific to inhibition of the binding of Aβ oligomer to PrP^C. Dextran on the other hand did not affect this binding. DSS also suppressed Aβ oligomer binding to cells expressing PrP^C but not to control cells. Furthermore, while incubation of mouse hippocampal slices with Aβ oligomers inhibited the induction of long-term potentiation（LTP）, simultaneous treatment with DSS restored the LTP. Since DSS has already been approved for use for patients with hypertriglyceridaemia, and its safety in humans has been confirmed, we propose further analysis of this drug as a candidate for AD treatment.

1O2-05 Endocytic pathology in astrocytes：dynein dysfunction disrupts Abeta clearance in astrocytes via disturbed endosome trafficking Kimura Nobuyuki¹，Okabayashi Sachi²，Ono Fumiko² ¹Section of Genetics and Molecular Biology, Department of Alzheimer’s Disease Research, National Center for Geriatrics and Gerontology，²The Corporation for Production and Research of Laboratory Primates A large number of studies suggest that endocytic disturbance is involved in Alzheimer’s disease（AD）pathogenesis, especially for β-amyloid protein（Aβ）pathology. Our previous studies showed that aging affects retrograde motor protein dynein in cynomolgus monkey brain, and dynein dysfunction reproduces age-dependent endocytic pathology, resulting in the accumulation of intracellular β-amyloid precursor protein（APP）and Aβ. These findings suggest that dynein dysfunction may be one of the responsible factors for age-dependent endocytis distrbance leading to AD pathogenesis. On the other hand, it remains unclear whether such age-dependent endocytic disturbance also occurs in glial cells. Here, we show that intracellular accumulation of enlarged endosomes occurs even in astrocytes of aged monkey brains, and we confirmed that Aβ accumulates in those enlarged endosomes. RNA interference studies demonstrated that dynein dysfunction reproduces astroglial endocytic pathology and disrupts Aβ clearance in astrocytes via disturbed endosome trafficking. Interestingly, dynein dysfunction did not affect Aβ uptake itself. These findings suggest that endocytic disturbance in astroglial cells may also be involved in age-dependent Aβ pathology.		1O2-06 Identification of domains of FUS required for the regulation of genes with conserved introns. Nakaya Tadashi Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University FUS is an RNA binding protein and known as a causative factor of Amyotrophic Lateral Sclerosis, ALS. Many mutations linked to familial cases of ALS have been identified in the C-terminal end, around its nuclear localization signal, and most of them abnormally localized in cytoplasm. Since FUS wild type strictly localizes in nucleus, it is believed that the mislocalization of FUS mutants is critical to pathogenesis of ALS. However, it is still unclear how mutations of FUS cause ALS. Because the normal function of FUS was not clear, which might be important to reveal the mechanism of FUS mutant effects, we employed HITS-CLIP and RNA-seq experiments to reveal the physiological function of FUS in neurons. We found that FUS bound to many RNAs through their introns and 3’UTR regions, which was observed in both human brains and mouse ES cell derived neurons. However, almost no significant difference of expressions of target RNAs was observed in FUS knock down neurons compared to control siRNA treated neurons. Thus far, it is still unclear what the normal function of FUS in neurons is. Among these FUS target introns, we found that FUS preferentially bound to introns conserved among species. Moreover, the expressions of these RNAs with conserved introns were affected significantly in FUS knock down neurons. Interestingly, many RNA binding protein-coding genes have these conserved introns, suggesting that FUS regulates the expressions of RNA binding proteins by regulating their RNA levels through their conserved introns. To reveal the molecular mechanism of this regulation, deletion constructs of FUS were utilized and the effects on target RNAs were analyzed. FUS has six domains including SYGQ-rich, Gly-rich, RNA recognition motif（RRM）, Arg-Gly-Gly-rich1（RGG1）, Zn-finger and RGG2. When these domains were deleted one by one, all of mutant localized to the nucleus, indicating that these domains were not involved in the regulation of its localization in neurons. In this presentation, I will show the results of further analyses and discuss how FUS regulates the expressions of RNAs with conserved introns.