ポリグルタミン病、ALS、脊髄小脳変性症、その他の神経変性疾患 Polyglutamine Diseases, ALS, SCD, Other Neurodegenerative Disorder
P3-1-223 Chorein結合タンパクの同定 Identification of binding partners of chorein ○塩川奈理1, 中村雅之1, 出口晃子1, 佐々木なつき1, 佐野輝1 ○Nari Shiokawa1, Masayuki Nakamura1, Akiko Deguchi1, Natsuki Sasaki1, Akira Sano1 鹿児島大学大学院医歯学総合研究科 精神機能病学分野1 Department of Psychiatry, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima Japan1 Chorea-acanthocytosis (ChAc) is a rare hereditary neurodegenerative disorder caused by loss-of-function mutations of the vacuolar protein sorting-associated protein13A (VPS13A) gene encoding a large protein, chorein. Chorein is expressed in various tissues, but its physiological function at molecular level is still unclear. In order to understand function of chorein, we investigated which proteins interact with chorein. Beta-adducin and beta-actin were co-immunoprecipitated with chorein from extracts of healthy human erythrocyte ghost membrane and human embryonic kidney 293 (HEK293) cells stably overexpressing chorein. We performed immunohistochemical analysis using HEK293 cells, which showed obvious colocalization of chorein with these proteins. Interestingly, the expression of beta-adducin is restricted to the brain and hematopoietic tissues. The pathological findings of ChAc are characterized by neurodegeneration in the brain and erythrocyte acanthocytosis. These results suggest that chorein may have functional relationships between beta-adducin and beta-actin and its dysfunction may lead to the brain pathology and the occurrence of acanthocytes in ChAc.	P3-1-224 ファール病と関連したリン酸トランスポーターPiT2のマウス脳内の局在に関する検討 A study on the localization in the mouse brain of type III sodium-dependent phosphate transporter 2 (PiT2) associated with Fahr's disease ○位田雅俊1, 入山真先1, 高木麻里1, 金子雅幸1, 保住功1 ○Masatoshi Inden1, Masaki Iriyama1, Mari Takagi1, Masayuki Kaneko1, Isao Hozumi1 岐阜薬大・薬・薬物治療1 Lab. Med. Therap. Mol. Therap., Gifu Pharm. Univ.1 Type III sodium-dependent phosphate transporters 1 and 2 (PiT1 and PiT2, respectively) are proteins encoded by the SLC20 A1 and 2 genes, respectively. The both broad distribution of PiT1 mRNA and PiT2 mRNA in mammalian tissues is compatible with the housekeeping maintenance of intracellular inorganic phosphate (Pi) homeostasis by absorbing Pi from interstitial fluid for normal cellular functions. Interestingly, mutations of SLC20A2 have been found in patients with idiopathic bilateral basal ganglia and cerebellar calcification (IBGC), also known as Fahr's disease. This suggests impaired Pi homeostasis in Fahr's disease. However, the localization of PiT2 in the normal brain has not been clarified yet. The aim of this study was to reveal the distribution of PiT2 expression in the mouse brain. The immunoblotting and immunohistochemical studies using a polyclonal antibody (Ab) and a monoclonal Ab showed that PiT2 immunopositivity was widely expressed throughout the brain. In the cellular type, it was intensely recognized in neurons co-localized with class III β-tubulin in the cerebral cortex, Purkinje cells co-localized with calbindin, and vascular endothelial cells. It was less intense in astrocytes identified with anti-GFAP Ab and least intense in microglia identified with anti-iba1 Ab. Our results indicate PiT2 plays an important role in the maintenance of cellular Pi homeostasis in neurons and endothelial cells. This is thought to be associated with the pathophysiology of Fahr's disease and suggests Fahr's disease primarily implicate the neuronal death.
P3-1-225 MRE11遺伝子異常は進行性ミオクローニックアタキシアを起こす Exome sequencing reveals a novel MRE11 mutation in a patient with progressive myoclonic ataxia ○宮本亮介1,2, 森野豊之1, 吉澤明生1, 宮崎由道2, 丸山博文1, 村上永尚2, 深田慶3, 和泉唯信2, 松浦伸也4, 梶龍兒2, 川上秀史1 ○Ryosuke Miyamoto1,2, Hiroyuki Morino1, Akio Yoshizawa1, Yoshimichi Miyazaki2, Hirofumi Maruyama1, Nagahisa Murakami2, Kei Fukada3, Yuishin Izumi2, Shinya Matsuura4, Ryuji Kaji2, Hideshi Kawakami1 広島大学原爆放射線医科学研究所分子疫学研究分野1, 徳島大学神経内科2, 大阪府立急性期・総合医療センター神経内科3, 広島大学原爆放射線医科学研究所放射線ゲノム疾患研究分野4 Department of Epidemiology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan1, Department of Clinical Neuroscience, Institute of Health Biosciences, Graduate School of Medicine, University of Tokushima, Tokushima, Japan2, Department of Neurology, Osaka General Medical Center, Osaka, Japan3, Department of Genetics and Cell Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan4 Objective, background: Progressive myoclonic ataxia is a clinical syndrome defined as progressive ataxia and myoclonus and infrequent seizures in the absence of progressive dementia. In contrast to the progressive myoclonic epilepsy, which can usually yield a specific diagnosis, a larger proportion of progressive myoclonic ataxia cases remain undiagnosed. The aim of this study was to clarify the molecular etiology of progressive myoclonic ataxia.Subject, methods: The patient was a 52-year-old female from consanguineous parents. She developed a jerky neck movement at age 9, which gradually expanded to her entire body. On physical examination at age 47, she showed generalized, spontaneous myoclonus that occurred continuously. She also presented mild limb and truncal ataxia. An electroencephalogram revealed no abnormalities. Brain MRI showed no atrophy in the cerebellum. Electrophysiological studies suggested myoclonus of a subcortical origin. For further evaluation, we performed exome sequencing.Results: We identified a novel homozygous missense mutation in the MRE11 gene (NM_005590:c.140C>T:p.A47V). Subsequently, we analyzed the expression of MRE11 and related proteins (RAD50 and NBS1) via Western blotting, and they showed a marked decrease.Conclusions: Mutations in the MRE11 gene have been known to cause an ataxia-telangiectasia-like disorder. Accumulating evidence has indicated that its wide phenotypic variations correspond to genotypic differences. Our case further expands this knowledge. Additionally, together with a recently reported case of an MRE11 mutation presenting as progressive myoclonic ataxia, it is suggested that MRE11 mutations can cause progressive myoclonic ataxia.	P3-1-226 CADASIL変異ではNOTCH3のトランスエンドサイトーシスが障害されている Transendocytosis is impaired in CADASIL mutant NOTCH3 ○渡邉明子1, 渡邊義久2, 田中雅樹2, 中川法正1, 水野敏樹1 ○Akiko Watanabe1, Yoshihisa Watanabe2, Masaki Tanaka2, Masanori Nakagawa1, Toshiki Mizuno1 京都府立医科大学　神経内科1, 京都府立医科大学　附属脳・血管系老化研究センター　基礎老化学部門2 Department of Neurology, Graduate School of Medical Science,Kyoto Prefectural University of Medicine1, Department of Basic Geriatrics,Research Institute for Neurological Disease and Geriatrics, Kyoto Prefectural University of Medicine2 Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is characterized by migraine, depression, recurring subcortical infarcts, and dementia. Its pathological vascular lesions are characterized by degeneration of vascular smooth muscle cells, strongly stained with antibody to the extracellular domain of NOTCH3 (N3ECD), and exhibit the presence of granular osmiophilic material (GOM) . Mutations in the human NOTCH3 gene cause CADASIL, but the pathogenesis of CADASIL has remained unclear. Recently, endocytosis of Notch ectodermal domain into the ligand expressing cells, called as transendocytosis, is considered to be critical for Notch activation. We hypothesized that the mutant NOTCH3 protein, particularly the ectodermal domain of NOTCH3 (N3ECD), may be refractory to degrade on the cell surface due to impaired transendocytosis. We established the co-culture system that HEK293 cells stably expressing one copy of tetracycline-regulated NOTCH3 were cultured with NOTCH3 ligand, Jagged1 (Jag1) expressing HEK293 cells. We obtained three main results: first, the wild N3ECD on the cell surface was degraded significantly more rapidly than C185R mutant when NOTCH3 cells were co-cultured with Jag1 expressing cells. Second, both the wild and mutant NOTCH3 expressing cells increased the HES1 expression by co-culturing with ligand expressing cells. Third, vesicles containing N3ECD were observed in Jag1 expressing cells. Vesicles of mutant N3ECD within the Jag1 expressing cells were significantly less than wild N3ECD. These results indicated that degradation process of mutant N3ECD on the cell surface is disturbed due to the impairment of the transendocytosis. Such disturbed proteolytic process of N3ECD on the surface of the vascular smooth muscle cells may contribute to a pathogenic mechanism of CADASIL.
P3-1-227 Choreinは、栄養飢餓状態におけるHEK293細胞において細胞骨格系蛋白と相互作用する Chorein interacts with cytoskeletal proteins on nutrient starvation in HEK293 cells ○佐々木なつき1, 中村雅之1, 塩川奈理1, 出口晃子1, 佐野輝1 ○Natsuki Sasaki1, Masayuki Nakamura1, Nari Shiokawa1, Akiko Deguchi1, Akira Sano1 鹿児島大院・医歯・精神機能1 Dept Psychiat, Univ of Kagoshima, Kagoshima1 Chorea-acanthocytosis (ChAc) is an autosomal recessive neurodegenerative disorder caused by loss of function mutations in the vacuolar protein sorting 13 homolog A (VPS13A) gene encoding chorein. The main neuropathological finding of ChAc is the degeneration of striatum. Although a deficiency in the function of chorein leads to ChAc, its detailed physiological role remain unclear.Recently, it was reported that the autophagy pathway had been implicated in several neurodegenerative disease such as Alzheimer disease, Huntington disease and Parkinson disease. Autophagy plays a protective role in preventing or reducing cytotoxicity by clearance of the toxic proteins.In the present study, the results of MTS cell viability assay, which is based on the cellular conversion of a tetrazolium salt into a formazan product, showed that in comparison with mock cells, HEK293 cells, stably expressing chorein, significantly preserved cell viability during nutrient deprivation. Moreover, co-immunoprecipitation and immunocytochemical analyses after nutrient deprivation revealed increased binding and colocalization of chorein and cytoskeletal proteins in HEK293 cells. These results suggest that chorein interacts with the cytoskeletal proteins and may play an important role in autophagic process, including in the protection against starvation-induced cell damages.	P3-1-228 Choreinは、酸化的リン酸化抑制剤で処理したHEK293細胞において細胞骨格蛋白と相互作用する Chorein interacts with cytoskeletal proteins in HEK293 cells treated with a chemical inhibitor of oxidative phosphorylation ○出口晃子1, 中村雅之1, 塩川奈理1, 佐々木なつき1, 佐野輝1 ○Akiko Deguchi1, Masayuki Nakamura1, Nari Shiokawa1, Natsuki Sasaki1, Akira Sano1 鹿児島大院・医歯・精神機能1 Dept Psychiat, Kagoshima Univ, Kagoshima1 Chorea-acanthocytosis (ChAc) is a rare disorder that is characterised by neurodegeneration at striatum and red cell acanthocytosis.ChAc is caused by loss of function mutations in the vacuolar protein sorting 13 homolog A (VPS13A) gene located on chromosome 9q21 encoding chorein. Although a deficiency in chorein function leads to degeneration of striatal neurons, its detailed physiological role remain unclear.It was recently considered that dysfunction of mitochondria play a central role in neurodegenerative diseases. We hypothesized that chorein is related to mitochondrial dysfunction.In the previous study, using a HEK293 cell line stably expressing chorein, we performed co-immunoprecipitation analysis, which revealed the interactions of chorein with cytoskeletal proteins. Immunocytochemically, chorein was colocalized with the cytoskeletal proteins and mitochondria in HEK293 cell. Additionally, the interactions were enhanced by treatment with the chemical inhibitor of oxidative phosphorylation. Taken together, these results suggest that chorein interacts with the cytoskeletal proteins and is involved in mitophagy.
P3-1-229 球脊髄性筋萎縮症モデルマウスに対するCDK阻害剤の効果 Cyclin-dependent kinase inhibitor mitigates motor neuron degeneration in spinal and bulbar muscular atrophy (SBMA) ○池中建介1, 勝野雅央1, 足立弘明1, 近藤直英1, 飯田円1, 中辻秀朗1, 南山誠1,2, 土井英樹1, 松本慎二郎1, 宮崎雄1, 藤内玄規1, 祖父江元1 ○Kensuke Ikenaka1, Masahisa Katsuno1, Hiroaki Adachi1, Naohide Kondo1, Madoka Iida1, Hideaki Nakatsuji1, Makoto Minamiyama1,2, Hideki Doi1, Shinjiro Matsumoto1, Yu Miyazaki1, Genki Tonai1, Gen Sobue11 名古屋大学大学院　医学系研究科　神経内科1, 国立長寿医療センター2 Dept Neurol, Univ of Nagoya, Nagoya1, National Center for Geriatrics and Gerontology, Obu, Japan2 Spinal and bulbar muscular atrophy (SBMA) is an adult-onset motor neuron disease caused by the expansion of a CAG trinucleotide repeat, which encodes a polyglutamine tract, in androgen receptor (AR) gene. Our previous study showed that the polyglutamine-expanded AR protein, the causative protein of SBMA, induces the expression of cell cycle regulators, such as cyclines, E2F1, and PCNA, and hyper-phosphorylation of pRb protein, in the motor neurons of a transgenic mouse model of SBMA (AR-97Q). The aim of this study is to test the effects of flavopiridol, a cyclin-dependent kinase inhibitor that inhibits cell cycle, in the mouse model of SBMA. Methods: Flavopiridol was intreventrically administered to male AR-97Q mice via an opmotic pump. The rotarod performance was assessed weekly using an Economex Rotarod, while the grip strength was measured with a Grip Strength Meter. The loss of motor neurons and gliosis were evaluated using immunohistochemistry. Results: Intraventricular administration of flavopridol suppressed the expression of cell cycle markers, inhibited BrdU uptake in motor neurons within the spinal anterior horn and brainstem. Rotarod performance, grip power, and life span of AR-97Q mice was also substantially improved by flavopiridol, whereas this agent showed no detectable effects on the motor performance of wild-type mice. Conclusions: The present study showed that cell cycle re-entry is associated with the pathogenesis of neurodegeneration in SBMA, and cyclin-dependent kinase inhibitor inhibits motor neuron degeneration by the polyglutamine-expanded AR protein.	P3-1-230 筋萎縮性側索硬化症のモデルマウスであるG93A マウスの運動神経変性はc-Abl 阻害により遅延する c-Abl Inhibition Delays Motor Neuron Degeneration in the G93A Mouse, an Animal Model of Amyotrophic Lateral Sclerosis ○勝又竜1, 石垣診祐1, 勝野雅央1, 河合香織1, 足立弘明1, 田中章景2, 祖父江元1 ○Ryu Katsumata1, Shinsuke Ishigaki1, Masahisa Katsuno1, Kaori Kawai1, Hiroaki Adachi1, Fumiaki Tanaka2, Gen Sobue1 名古屋大学医学部神経内科1, 横浜市大病院神経内科2 Department of Neurology, Nagoya University Graduate School of Medicine1, Department of Neurology and Stroke Medicine , Yokohama City University Graduate School of Medicine2 Background: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive death of motor neurons. Although the pathogenesis of ALS remains unclear, several cellular processes are known to be involved, including apoptosis. A previous study revealed the apoptosis-related gene c-Abl to be upregulated in sporadic ALS motor neurons.Methodology/Findings: We investigated the possibility that c-Abl activation is involved in the progression of ALS and that c-Abl inhibition is potentially a therapeutic strategy for ALS. Using a mouse motor neuron cell line, we found that mutation of Cu/Zn-superoxide dismutase-1 (SOD1), which is one of the causative genes of familial ALS, induced the upregulation of c- Abl and decreased cell viability, and that the c-Abl inhibitor dasatinib inhibited cytotoxicity. Activation of c-Abl with a concomitant increase in activated caspase-3 was observed in the lumbar spine of G93A-SOD1 transgenic mice (G93A mice), a widely used model of ALS. The survival of G93A mice was improved by oral administration of dasatinib, which also decreased c-Abl phosphorylation, inactivated caspase-3, and improved the innervation status of neuromuscular junctions. In addition, c-Abl expression in postmortem spinal cord tissues from sporadic ALS patients was increased by 3-fold compared with non-ALS patients.Conclusions/Significance: The present results suggest that c-Abl is a potential therapeutic target for ALS and that the c-Abl inhibitor dasatinib has neuroprotective properties in vitro and in vivo.
P3-1-231 Immunohistochemical distribution of the Huntingtin-associated protein 1 (HAP1) in the spinal cord of adult rat ○MD Islam1, Ryutaro Fujinaga1, Mir R. Jahan1, Akie Yanai1, Keiji Kokubu1, Naoto Hayasaka1, Ren Yonetani1, Koh Shinoda1 Divsion of Neuroanatomy, Yamaguchi Uni. Grad. Sch. of Medicine1 Huntingtin-associated protein 1 (HAP1) is a neural huntingtin interactor that is widely expressed as a core molecule of the stigmoid body (STB) and has putative protective functions against neurodegeneration. Although expression of STB/HAP1 has been well described in the brain, little is known about their presence in the spinal cord, which is another important target for neurodegenerative diseases. The purpose of the present study is to clarify the detail immunohistochemical distribution of STB/HAP1 in the spinal cord of rat. We used frozen sections of all segments of the spinal cord fixed with 4% paraformaldehyde of adult Wistar male rat. Immunoperoxidase or immunofluorescence staining was performed using antibody against HAP1. Most of the HAP1- immunoreacive (ir) cells were present in the gray matter of the spinal cord, while no HAP1-ir cells was detected in white matter except lateral spinal nucleus. HAP1-ir cells were abundantly and densely expressed in the posteromarginal nucleus (lamina I) and substantia gelatinosa (lamina II) of the dorsal horn, sympathetic preganglionic neurons and parasympathetic preganglionic neurons of lamina VII, and around the central canal (lamina X) ,whereas in the intermediate zone (lamina III to VIII) HAP1-ir cells were sparsely scattered. In contrast, no HAP1-ir cells were found in the motor neurons of the lamina IX. Almost all the HAP1-ir cells belonged to neurons but not in glial cells, as coexpressed with NeuN. Majority of the positive cells showed clear HAP1-ir STB in the diffusely immunoreactive cytoplasm, while the few others showed only diffuse HAP1 immunoreactivity in the cytoplasm with no clear STB. Our present study suggests that sensory and autonomic neurons in the spinal cord should be stable against neurodegenration due to putative STB/HAP1 protectivity, whereas, due to absence of STB/ HAP1spinal motorneurons might be vulnerable to neurodegenerative diseases	P3-1-232 筋萎縮性側索硬化症におけるガレクチン1の二面的作用 Dual effects of Galectin-1 in amyotrophic lateral sclerosis ○小早川優子1, 作見邦彦1, 中別府雄作1 ○Yuko Kobayakawa1, Kunihiko Sakumi1, Yusaku Nakabeppu1 九大・生医研・脳機能制御学1 Div. of Neurofunc. Genomics, Med. Inst. of Bioreg., Kyushu Univ.1 Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease which shows progressive muscle weakness associated with degeneration of motor neurons. Its molecular mechanism is still largely unclear. Galectin-1, a member of the β-galactoside-binding lectin family, expresses in various tissues and has multiple functions such as cell proliferation, cell adhesion, tumor metastasis and apoptosis of activated T cell. It has been shown that reduced form of Galectin-1 exists as a homodimer while its oxidized form exists as a monomer, and each form exerts different effects on cells. The oxidized Galectin-1 is known to promote the axonal regeneration after axotomy of peripheral nerve. It has been reported that Galectin-1 is accumulated in axonal spheroids of spinal cord, in both sporadic and SOD1 mutant familial ALS patients. Additionally, the oxidized Galectin-1 has been shown to be neuroprotective in a mutant SOD1-Tg mouse model of ALS. We thus examined roles of Galectin-1 in the pathogenesis of ALS by cross-breeding the ALS model mice (SOD1G93A) with Galectin-1 null mice (Lgals1-/-). First, we elucidated the expression pattern of Galectin-1 in SOD1G93A mice. Galectin-1 is accumulated in axonal spheroids long before disease onset. While after the onset, astrocytes around axons of motor neurons express high level of Galectin-1. We then evaluated effects of Galectin-1 knockout in the SOD1G93A mice. SOD1G93A/Lgals1-/- mice exhibit significantly delayed onset of motor dysfunction in comparison to SOD1G93A/Lgals1+/+ mice. However, disease progression was accelerated in SOD1G93A/Lgals1-/- mice after the onset of motor dysfunction, and there was no difference in survival curve between SOD1G93A/Lgals1-/- mice and SOD1G93A/Lgals1+/+ mice.We thus suggest that Galectin-1 accumulated in the axons of motor neurons promotes neuronal degeneration, however, Galectin-1 expressed in astrocytes at the symptomatic stage is neuroprotective.
P3-1-233 大脳皮質神経細胞におけるTDP-43及びFUSのtranscriptomeプロファイルの比較検討 A comparative analysis of the transcriptome profiles of TDP-43 and FUS in the primary cortical neurons ○本田大祐1, 井口洋平1, 藤岡祐介1, 増田章男2, 渡辺宏久1, 勝野雅央1, 大野欽司2, 石垣診祐1, 祖父江元1 ○Daiyu Honda1, Yohei Iguchi1, Yusuke Fujioka1, Akio Masuda2, Hirohisa Watanabe1, Masahisa Katsuno1, Kinji Ohno2, Shinsuke Ishigaki1, Gen Sobue1 名古屋大学大学院医学系研究科神経内科学1, 名古屋大学大学院医学系研究科附属 神経疾患・腫瘍分子医学研究センター2 Department of Neurology Nagoya University Graduate School of Medicine1, Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine2 TAR DNA-binding protein (TDP-43) and FUS have been linked to both familial and sporadic ALS and its nascent disease entity, frontotemporal lobar degeneration (FTLD). Both TDP-43 and FUS are RNA binding proteins which function in transcription, RNA splicing, and RNA transport, and share similar clinicopathological features of ALS/FTLD in which TDP-43 and/or FUS are associated. To obtain pathophysiological RNA metabolism cascade originated from TDP-43 and FUS, we compared the TDP-43-regulating and Fus-regulating profiles of differential gene expression and alternative splicing in primary cortical neurons. We introduced two different shRNAs against either TDP-43 or FUS into primary cortical neurons using lentivirus. The profiles of gene expression and alternative splicing events were analyzed by exon-sensitive microarray. The scattered plot analysis revealed that both gene expression and alternative splicing profiles of genes regulated by FUS and TDP-43 were well correlated. We obtained 183 and 204 genes whose expression levels were significantly changed in FUS-depleted neurons and TDP-43-depleted neurons, respectively (t-test, p-value<0.05 and FC>1.5). Among them, the expression levels of 51 genes were concordantly changed in both groups. In the comparison of alternative splicing events, 428 exons were significantly altered in FUS-depleted neurons, whereas 674 exons were significantly altered in TDP-43 depleted neurons (t-test, p-value<0.05 and FC>1.5). 61 exons were concordantly altered in both FUS-depleted and TDP-43 depleted neurons. Profile comparison with Cugbp1, another RNA binding protein revealed that there was no profile correlation between Cugbp1-depleted neurons and FUS-depleted or TDP-43-depleted neurons. The common downstream RNA targets for both TDP-43 and FUS would give us important insights into ALS/FTLD pathogenesis.

上部に戻る

前に戻る