一般演題（ポスター）

一般演題（ポスター） Parkinson’s Disease・Neuronal Degeneration・Muscle Disorders

3P-40 PUFA-derived lipid peroxide enhances alpha-synuclein toxicity through perturbation of autophagy system Maruyama Wakako，Shamoto-Nagai Masayo，Naoi Makoto Dept of Health Nutr Facult. Psycol. Physical Sci. Aichi Gakuin Univ. Docosahexaenoic acid（DHA）is a major fatty acid composition of the neuronal membrane. DHA is α-Synuclein αSyn）has been proposed to be associated with the pathogenesis of Parkinson disease（PD）. and stabilizes conformation of αSyn in α-helical structure. The cytotoxicity of αSyn depends on its higher structure, including momomeric, oligomeric and aggregated forms. DHA is known as a potent anti-oxidant but simultaneously, it is oxidized and produces cytotoxic lipid-radicals. This paper reports that DHA peroxidation modified αSyn and induced the oligomerization and facilitated the amyloidogenesis in vitro. DHA induced cell death with increased αSyn adduct with N-acyl product from DHA peroxidation and cell death in SH-SY5Y cells overexpressing αSyn. Using this model, the proteolysis system was studied. αSyn overexprssion itself activated autophagy system. In addition, DHA-derived lipid-derived oxidative stress disturbed autophagy-lysosomal fusion and as a result, the accumulation of abnormal proteins in the cells was observed. This finding indicates that oxidation of DHA which is rich in neuronal membrane may enhance the toxic proteins by inhibiting proteolysis system.		3P-41 Analysis of contextual fear memory and hippocampal CREB phosphorylation in 1-methyl-4-phenyl-1, 2, 3, 6, tetrahydropyridine（MPTP）-induced mouse model of Parkinson’s disease Kinoshita Ken-ichi^1,2，Muroi Yoshikage²，Ishii Toshiaki^1,2 ¹Det. of Pathogenetic Vet. Sci., Gifu Univ. Utd Grad Sch of Vet. Sci.，²Det. of Basic Vet. Med., Obihiro Univ. of Agri. and Vet. Med. In order to clarify the mechanism of the cognitive deficits in Parkinson’s disease（PD）, we investigated the mechanism of cognitive deficits in MPTP-induced PD model mice（PD mice）. PD mice were produced by MPTP（four injections at a single dose of 20 mg/kg every 2 h, i.p.）, which destroys specifically the nigrostriatal dopaminergic neurons. The number of tyrosine hydroxylase positive cells in the substantia nigra pars compacta was significantly decreased in PD mice. We evaluated the cognitive function of PD mice using the contextual fear conditioning test. In the test, we conducted using a weak unconditioned stimulus（US）（1 mA/2 s, single）or an intense US（2 mA/2 s, twice）, and evaluated fear consolidation, reconsolidation and extinction. Under the weak US in reconsolidation and extinction tests, there were no significant differences in the freezing rates between control and PD mice. When we conducted the tests under the intense US, memory reconsolidation of PD mice normally occurred, but the memory of PD mice was attenuated earlier than the control mice by brief exposures to CS（3 min）every 24 h. In the extinction test, the PD mice showed a significant reduction in freezing rate earlier than the control mice. Next, we examined the expression level of the phosphorylated CREB（p-CREB）that is critical for fear memory formation. After an extinction training under the intense US, PD mice showed significant reduction in the number of p-CREB positive cells in hippocampal dentate gyrus（DG）. The p-CREB expression was observed specifically in immature cells（Doublecortin）but not in mature cells（NeuN）. These results suggest that the enhancement of memory extinction observed in PD mice may cause from a decrease in the p-CREB positive immature cells in hippocampal DG.

3P-42 Expression of the huntingtin-associated protein 1-immunoreactive stigmoid body and its morphological relationship with androgen receptor in the spinal cord of adult rat Islam Md Nabiul，Fujinaga Ryutaro，Takeshita Yukio，Yanai Akie，Jahan Mir Rubayet，Wroblewski Greggory J，Shinoda Koh Div. of Neuroanatomy, Dept. of Neuroscience, Yamaguchi University Grad. School of Medicine Huntingtin-associated protein 1（HAP1）is a neural huntingtin interactor and is considered to be a determinant marker of the stigmoid body（STB）. STB/HAP1 has putative protective functions against some neurodegenerative diseases（STB/HAP1 protection hypothesis）. Although the expression of STB/HAP1 has been well described in the brain, little is known about its presence in the spinal cord which is also vulnerable to neurodegenerative diseases like spinal and bulbar muscular atrophy（SBMA）that is caused by mutation of androgen receptor（AR）. We immunohistochemically determined the distribution of STB/HAP1 and its morphological relationship with AR in the spinal cord of adult Wistar rats of both sexes in light,fluorescence, and electron microscopy as well as western blotting. In this study, almost all STB/HAP1-immunoreactive（ir）cells belonged to neurons, but not glial cells, as indicated by their co-expression with NeuN but not with GFAP, Iba1 or Olig2. About 90% of neurons in the lamina I-III, sympathetic and parasympathetic preganglionic cells, as well as in the lamina X, expressed STB/HAP1. In addition, about 50% of neurons in lamina IV and V expressed STB/HAP1, whereas STB/HAP1-ir cells were relatively sparse（15-30%）in lamina VI, VII and VIII. In contrast, no STB/HAP1-ir cells were found in the motoneurons of the lamina IX. Our present study suggests that STB/HAP1 in the spinal cord might play an important role in diverse spinal sensory and autonomic functions. Sensory and autonomic neurons in the spinal cord should be stable against stressful conditions as inducing neurodegeneration, due to putative STB/HAP1 protectivity, whereas the motoneurons might be vulnerable to such stresses due to the absence of STB/HAP1 in lamina IX. Interestingly, more than 80% of AR-ir cells in the dorsal horn or around the central canal contained STB/HAP1, but AR-ir cells in the ventral horn motoneurons were devoid of STB/HAP1. Our current results strongly support STB/HAP1 protection hypothesis in-vivo and might explain why the spinal motoneurons are major target in some neurodegenerative diseases including SBMA.		3P-43 TDP43 recognizes and transports G4-containing mRNAs into neurites for local translation Ishiguro Akira¹，Kimura Nobuyuki² ¹Research Center for Micro-Nano Technology, University of Hosei，²Section of Cell Biology and Pathology, Department of Alzheimer’s Disease Research, Center for Development of Advanced Medicine for Dementia, National Center for Geriatrics and Gerontology Local protein synthesis within neurons is an essential mechanism for the establishment and conservation of synaptic plasticity, neural activities, and cell polarity. RNA-binding proteins（RBPs）are considered to play key roles in transport and distribution of specific mRNAs into target sites for the local protein synthesis. At present, however, little is known on the mechanism how these RBPs mediate selective transport of specific mRNAs into the target area.TDP-43（TAR DNA-binding 43 kDa protein encoded by TARDBP）, a ubiquitously expressed RBP in various tissues, contains two RNA recognition motifs and a Gly-rich domain, and forms a homo-dimer in normal cells under physiological conditions. In neurons of patients with amyotrophic lateral sclerosis（ALS）, frontotemporal lobar degeneration（FTLD）or some neurodegenerative disorders, TDP-43 exists as a major component of the ubiquitin-positive inclusions, the pathological hallmarks of neuron diseases.In order to identify the binding target RNAs and the recognition sequences of TDP-43, we performed SELEX（systematic evolution of ligands by exponential enrichment）screening. The collection of RNA sequences contained G-quadruplex structure, which is formed from the stacking of two or more guanine tetrads. G-quadruplex structures have been identified in 3’-UTR of approximately 30% of the well-known dendritic mRNAs. We found that TDP-43 recognizes G-quadruplex containing mRNAs and transports them up to neurites for local translation. Furthermore we demonstrate that a TDP-43 with ALS-linked mutation is unable to co-localize with target RNAs. Finally TDP-43 was found to bind the G-quadruplex-forming RNA encoded by ALS and FTLD associated GGGGCC hexanucleotide repeat expansion of the C9orf72 gene. Taken together we propose that TDP-43 plays a key role in intracellular trafficking of G-quadruplex-containing mRNAs for the local protein synthesis in neurite.

3P-44 Cellular analysis of aberrant proteins derived from expanded GGGGCC repeat associated with ALS Takitani Mika，Kino Yoshihiro，Yamamoto Yoji，Kimura Misaki，Miyoshi Junko，Satoh Jun-ichi Dept Bioinformatics, Meiji Pharmaceutical Univ. It has recently been recognized that amyotrophic lateral sclerosis（ALS）and frontotemporal dementia share common pathological and genetic features. For example, pathological inclusions containing TDP-43 are found in a majority of ALS and a subset of FTD cases. In 2011, an expansion of a GGGGCC repeat in the intron 1 of the C9orf72 gene was identified in autosomal dominant ALS-FTD families. This mutation is thought to be the most frequent cause of familial ALS. Interestingly, the pathology of ALS with C9orf72 expansion shows intracellular inclusions with and without TDP-43. Recent reports indicate that the repeat expansion leads to RNA toxicity associated with ribonuclear inclusions and protein toxicity of dipeptide repeat produced from the expanded RNA through repeat associated non-ATG translation（RAN translation）. However, it is still unclear how these abnormal products cause diseases and which of these products is the most responsible for the pathogenesis. Here, we tried to establish an experimental system of GGGGCC repeat expression in cultured cells. While short GGGGCC repeats could be maintained stably in E. coli, （GGGGCC）50 was highly unstable and even plasmids from a single bacterial colony harbored variable lengths of the repeat. We determined the condition that stabilizes long repeat tracts. We inserted（GGGGCC）51 into the coding region downstream of EGFP with different reading frames. Upon transfection of these constructs into mammalian cells, we observed differential intracellular localization and aggregation properties depending on the reading frame of the repeat tract. Some aggregates were positive for ubiquitin or p62. Short Gly-Ala repeat protein alone did not show apparent aggregation, while it was detected in protein aggregates when co-expressed with a long repeat tract. These results not only reproduced some of recent results by others but also provided novel information on the properties of repeat-derived products. Our repeat expression system can be used for various cellular analyses, including the relationship between RNA foci formation and RAN translation and identification of dipeptide-associated proteins.		3P-45 Adenovirus-induced neuronal TDP-43 and FUS aggregates demonstrated by time-lapse imaging Watabe Kazuhiko¹，Ishii Tomohiro^1,3，Kawakami Emiko¹，Yanagisawa Hiroko¹，Akiyama Keiko¹，Endo Kentaro²，Misawa Hidemi³ ¹Laboratory for Neurodegenerative Pathology, Tokyo Metropolitan Institute of Medical Science，²Center for Basic Technology Research, Tokyo Metropolitan Institute of Medical Science，³Dept. of Pharmacology, Keio University Faculty of Pharmacy Formation of TDP-43- or FUS-positive cytoplasmic aggregates in neuronal and glial cells is one of the pathological hallmarks of amyotrophic lateral sclerosis（ALS）. We have previously demonstrated that proteasome inhibition enhanced adenovirus-induced neuronal cytoplasmic aggregate formation of TDP-43 and FUS in vitro and in vivo, suggesting that impairment of protein degradation pathways accelerates formation of TDP-43 and FUS-positive aggregates in ALS. However, the relationship between the cytoplasmic aggregate formation and the cell death remains unclear. In this study, we performed time-lapse imaging analysis of neuronal cells infected with adenoviruses encoding TDP-43 and FUS cDNAs under conditions of proteasome inhibition. Rat neural stem cell lines stably transfected with EGFP or Sirius under the control of tubulin beta III（TBB3p）, HB9, choline acetyl transferase（ChAT）, or vesicular acetylcholine transporter（VAChT）promoter were differentiated in the presence of retinoic acid with or without smoothened agonist SAG, followed by infection of neurogenin-2, Islet-1 and Lhx3 adenoviruses in case of motoneuron differentiation. The differentiated neuronal/motoneuronal cells were then infected with adenoviruses encoding DsRed-tagged human wild type and C-terminal fragment（CTF）TDP-43 or mutant P525L FUS in the presence of proteasome inhibitor MG-132 or an adenovirus encoding shRNA for proteasome PSMC1. Time lapse imaging analysis revealed growing DsRed-positive cytoplasmic aggregates in the infected neuronal/motoneuronal cells followed by the cell collapse within 72 hours. Released cytoplasmic aggregates composed of WT and CTF TDP-43 remained insoluble in the culture media over 30 hours of the time course. We are also attempting to develop time lapse imaging of cell to cell spreading of cytoplasmic aggregates.

3P-46 Astrocyte-derived TGF-β1 accelerates disease progression in ALS mice by regulating the neuroprotective inflammatory response of microglia and T cells Yamanaka Koji，Endo Fumito，Komine Okiru，Jin Shijie，Watanabe Seiji Research Institute of Environmental Medicine, Naogya University Neuroinflammation, consisted of both neuroprotective and neurotoxic reactions mediated by activated glial cells and infiltrated immune cells, is involved in the pathomechanism of amyotrophic lateral sclerosis（ALS）. However, the cytokines which regulate neuroprotective inflammatory response in ALS are not clarified. This study aims to elucidate the roles of TGF-β1, the elevated levels of which has been observed in the cerebrospinal fluid of ALS patients, in the context of neuroinflammation of ALS. We found that TGF-β1 levels were elevated in astrocytes of both murine and human ALS. By crossbreeding of SOD1^G93A and GFAP-TGF-β1 mice, astrocyte-specific overproduction of TGF-β1 in SOD1^G93A mice accelerated disease progression with reduced IGF-I production in deactivated microglia and fewer infiltrated T cells with a deregulated IFN-γ/IL-4 balance. Moreover, astrocyte-specific deletion of mutant SOD1 in loxSOD1^G37R mice resulted in slowing disease progression with a decreased level of TGF-β1 in astrocytes. Pharmacological administration of TGF-β signaling inhibitor after onset extends survival time of SOD1^G93A mice. In summary, we identify astrocytic TGF-β1 as a detrimental factor in accelerating disease progression of ALS through inhibiting the neuroprotective inflammatory response by microglia and T cells. Inhibition of TGF-β signaling in these cells may represent a novel therapeutic target for slowing disease progression of ALS.