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Establishment of iPS cells from amyotrophic lateral sclerosis model mice and motor neuronal differentiation
Abstracts / Neuroscience Research 71S (2011) e108–e415
P2-s07 Establishment of iPS cells from amyotrophic lateral sclerosis model mice and motor neuronal differentiation
Kenichi Komatsu 1 , Haruhisa Inoue 2,3 , Takayuki Kondo 1 , Shiho Kitaoka 2,3 , Kazutoshi Takahashi 2,3 , Shinya Yamanaka 2 , Ryosuke Takahashi 1 1
Dept Neurol, Kyoto Univ, Kyoto, Japan 2 Center for iPS Cell Research and Application, Kyoto Univ, Kyoto, Japan 3 JST-CREST, Tokyo, Japan
Subject: To unravel pathogenesis of amyotrophic lateral sclerosis (ALS), methods using induced pluripotent stem cells (iPS cells) are promising. We establish iPS cells from ALS model mice, mutant superoxide dismutase1 (SOD1) transgenic mice, induce neural differentiation and unravel pathogenesis of ALS. Methods: We introduced known four factors (Oct3/4, Sox2, Klf4, c-Myc) into mouse embryonic fibroblasts obtained from crossbreeding of SOD1G93A mice with Nanog-GFP-IRES-Puror mice using retroviral vectors and cultured them on SNL feeder cells. We picked ES cell-like colonies, cultured them and confirmed establishment of iPS cells using ES cell markers. Then we induced directed differentiation using retinoic acid and Smoothened agonist and confirmed motor neuronal identity by immunocytochemistry. Results: We established iPS cells from mutant SOD1 mice and control mice and confirmed motor neuronal differentiation. Conclusions: These results indicate that iPS cells from ALS model mice possess properties of ES cells with mutant SOD1 and may contribute to establishment of ALS model(s) in vitro. Research fund: Health and Labour Sciences Research Grants. doi:10.1016/j.neures.2011.07.842
P2-s08 Distinct mechanisms between divalent and monovalent MuSK antibodies are involved with dysfunction of MuSK leading to myasthenia gravis Shuuichi Mori , Sachiho Kubo, Shigeru Miyazaki, Kazuhiro Shigemoto
Yamada, Tsuyoshi
Dep. Geriatric Med., Tokyo Metro. Inst. of Gerontology, Itabashi, Japan Currently, it has been demonstrated that a variable proportion of patients with myasthenia gravis (MG) who lacked antibodies against acetylcholine receptor (AChR) have antibodies against muscle-specific kinase (MuSK). Although it has been thought that disruption of MuSK functions by the antibodies causes MG, the detailed mechanism by which MuSK antibodies interfere with MuSK functions remains unclear. Recently, it was shown that MuSK-IgG antibodies could cause the internalization of MuSK from cell surface. However, it may be difficult that this mechanism simply apply to all cases because a major subclass of the antibodies in humans is a functionally monovalent antibody and unable to crosslink identical antigens. Here, we generated the monovalent Fab fragments of divalent MuSK-IgG antibodies to investigate the mechanism of action of the antibodies in the interference with MuSK functions in vitro. When added to cultured C2C12 myotubes, both the divalent and the monovalent antibodies from the rabbits affected with MuSK-MG could interfere with the agrin-induced AChR clustering. In addition, the divalent antibodies induced the phosphorylation of MuSK and accelerated downregulation of Dok-7, an essential intracellular binding protein of MuSK, in the absence of agrin, and the monovalent antibodies inhibited the agrin-induced phosphorylation of MuSK. Interestingly, the divalent antibodies from one of the MuSK-MG patients could not induce the MuSK phosphorylation despite their abilities to inhibit the agrininduced AChR clustering, and their modes of action resembled those of the monovalent antibodies. These results demonstrate that the distinct mechanisms between two types of antibodies are involved with the dysfunction of MuSK, and the strong possibility that the functionally monovalent antibodies actually exist in the patients with MuSK-MG. doi:10.1016/j.neures.2011.07.843
e195
Multiple acyl-CoA dehydrogenation deficiency (MADD) is an autosomal recessive disease affecting amino acid, fatty acid, and choline metabolism and is a common genetic defect responsible for lipid-storage myopathy. Most forms of MADD are caused by a deficiency of electron transfer flavoprotein (ETF) or ETF dehydrogenase (ETFDH). However, its molecular feature has not been found uniformly in previous reports of Chinese patients. A large cohort of 56 late-onset MADD patients from 51 unrelated pedigrees in the southern China was recruited to investigate clear correlation between clinical phenotype and molecular genetic basis. All exons of ETFA, ETFB and ETFDH including the intron–exon boundaries, and 5 and 3 untranslated regions were directly sequenced. ETFDH deficiencies affected 94.1% (48/51) of the pedigrees. ETFDH-c.250G>A is the most common mutation, representing a high allelic frequency of 83.3% (80/96). Carrier frequency of c.250G>A is estimated to 1.35% (7/520) in the normal population. A significant reduced expression of ETFDH was identified in muscle of ETFDH-deficient patients. ETFDH deficiency is a major cause of riboflavin-responsive MADD in the southern China and c.250G>A is an important mutation that could be employed as a fast and reliable screening method. Research fund: A key program of scientific research of Fujian Medical University (2009D064), a program for Innovative Research Teams in Science (FMU-RT002), Fuzhou, and a grant from Huashan Hospital for the special professorship of Fudan University, Shanghai (to Wu Z.Y.), China. doi:10.1016/j.neures.2011.07.844
P2-s10 RGMa modulates T cell responses and is involved in autoimmune encephalomyelitis Rieko Muramatsu 1,2 , Takekazu Kubo 3 , Masahiro Mori 4 , Yuka Fujita 1,2 , Satoshi Kuwabara 4 , Toshihide Nakamura 1,2 , Yuki 1,2,3 Yamashita 1
Dep. of Mol. Neurosci., Grad. Sch. of Med., Osaka Univ., Osaka, Japan 2 JSTCREST 3 Dep. of Neurobiol., Grad. Sch. of Med., Chiba University, Chiba, Japan 4 Dep. of Neurol., Grad. Sch. of Med., Chiba University, Chiba, Japan Repulsive guidance molecule-a (RGMa) is a membrane-bound protein that was originally identified as an axon guidance molecule in the development of visual system. Although RGMa is recognized as having a crucial role in the nervous system, the role of RGMa in the immune system is unclear. In this study, we found that RGMa in dendritic cells increase clinical severity of experimental autoimmune encephalomyelitis (EAE) mouse, an animal model of multiple sclerosis (MS). RGMa is expressed in bone marrow-derived dendritic cells (BMDCs) and that CD4+ T cells express receptors for RGMa. We found that RGMa binding to CD4+ T cells led to Rap1 activation and increased adhesion to intracellular adhesion molecule-1 (ICAM-1). Further, treatment with neutralizing antibodies to RGMa prevented mouse myelin oligodendrocyte glycoprotein (MOG)-induced EAE and reduced invasion by inflammatory cells. Adoptive transfer of stimulated BMDCs with RGMa-knockdown or stimulated CD4+ T cells from RGMa-specific antibody-treated EAE mice lead to moderately reduced clinical severity of EAE. CD4+ T cells from mice treated with RGMa-specific antibody had a diminished MOG-specific proliferative response and reduced Interferon-␥ Interleukin-2 (IL-2), IL-4, and IL-17 secretion. T cell response of purified peripheral blood mononuclear cells isolated from individuals with MS also reduced by the treatment with RGMa-specific antibodies. These results suggest that RGMa-specific antibody suppresses the T cell response to antigens; thus, we consider RGMa a promising molecular target for treating MS. doi:10.1016/j.neures.2011.07.845
P2-s11 Prostaglandin I2 -IP signaling promotes migration of oligodendrocyte precursor cell by a mechanism dependent on cAMP Chisato Takahashi 1,2 Yamashita 1,2
, Rieko
Muramatsu 1,2 , Toshihide
1
P2-s09 Molecular analysis of 51 unrelated pedigrees with late-onset multiple acyl-CoA dehydrogenation deficiency (MADD) in the southern China confirmed the most common ETFDH mutation and high carrier frequency of c.250G>A Zhi-Qiang Wang 1 , Xue-Jiao Chen 1 , Shen-Xing Murong 1 , Ning Wang 1 , Zhi-Ying Wu 1,2 1 Department of Neurology and Institute of Neurology, First Affiliated Hospital, Fujian Medical University 2 Department of Neurology and Institute of Neurology, Huashan Hospital, Institutes of Brain Science and State Key Laboratory of Medical Neurobiology, Shanghai Medical College, Fudan University
Dep. of Mol. Neurosci., Grad. Sch. of Med., Osaka Univ., Osaka, Japan 2 Japan Science and Technology Agency, CREST, Tokyo, Japan Oligodendrocytes regulate myelination through axon–oligodendrocyte interaction in the central nervous system (CNS). Formation of myelin sheath around axon enables rapid impulse propagation and protection against axonal damage. Thus, understanding of the mechanisms in CNS myelination and promotion of myelination contribute to recovery of neuronal function in demyelinating disorders, such as multiple sclerosis. Myelin repair after CNS disorders is promoted by migration of oligodendrocyte precursor cells (OPCs); however, the molecular mechanism of OPCs migration remains unclear. Here, we show that prostaglandin I2 (PGI2 ) is a promoter
P2-s07 Establishment of iPS cells from amyotrophic lateral sclerosis model mice and motor neuronal differentiation
Kenichi Komatsu 1 , Haruhisa Inoue 2,3 , Takayuki Kondo 1 , Shiho Kitaoka 2,3 , Kazutoshi Takahashi 2,3 , Shinya Yamanaka 2 , Ryosuke Takahashi 1 1
Dept Neurol, Kyoto Univ, Kyoto, Japan 2 Center for iPS Cell Research and Application, Kyoto Univ, Kyoto, Japan 3 JST-CREST, Tokyo, Japan
Subject: To unravel pathogenesis of amyotrophic lateral sclerosis (ALS), methods using induced pluripotent stem cells (iPS cells) are promising. We establish iPS cells from ALS model mice, mutant superoxide dismutase1 (SOD1) transgenic mice, induce neural differentiation and unravel pathogenesis of ALS. Methods: We introduced known four factors (Oct3/4, Sox2, Klf4, c-Myc) into mouse embryonic fibroblasts obtained from crossbreeding of SOD1G93A mice with Nanog-GFP-IRES-Puror mice using retroviral vectors and cultured them on SNL feeder cells. We picked ES cell-like colonies, cultured them and confirmed establishment of iPS cells using ES cell markers. Then we induced directed differentiation using retinoic acid and Smoothened agonist and confirmed motor neuronal identity by immunocytochemistry. Results: We established iPS cells from mutant SOD1 mice and control mice and confirmed motor neuronal differentiation. Conclusions: These results indicate that iPS cells from ALS model mice possess properties of ES cells with mutant SOD1 and may contribute to establishment of ALS model(s) in vitro. Research fund: Health and Labour Sciences Research Grants. doi:10.1016/j.neures.2011.07.842
P2-s08 Distinct mechanisms between divalent and monovalent MuSK antibodies are involved with dysfunction of MuSK leading to myasthenia gravis Shuuichi Mori , Sachiho Kubo, Shigeru Miyazaki, Kazuhiro Shigemoto
Yamada, Tsuyoshi
Dep. Geriatric Med., Tokyo Metro. Inst. of Gerontology, Itabashi, Japan Currently, it has been demonstrated that a variable proportion of patients with myasthenia gravis (MG) who lacked antibodies against acetylcholine receptor (AChR) have antibodies against muscle-specific kinase (MuSK). Although it has been thought that disruption of MuSK functions by the antibodies causes MG, the detailed mechanism by which MuSK antibodies interfere with MuSK functions remains unclear. Recently, it was shown that MuSK-IgG antibodies could cause the internalization of MuSK from cell surface. However, it may be difficult that this mechanism simply apply to all cases because a major subclass of the antibodies in humans is a functionally monovalent antibody and unable to crosslink identical antigens. Here, we generated the monovalent Fab fragments of divalent MuSK-IgG antibodies to investigate the mechanism of action of the antibodies in the interference with MuSK functions in vitro. When added to cultured C2C12 myotubes, both the divalent and the monovalent antibodies from the rabbits affected with MuSK-MG could interfere with the agrin-induced AChR clustering. In addition, the divalent antibodies induced the phosphorylation of MuSK and accelerated downregulation of Dok-7, an essential intracellular binding protein of MuSK, in the absence of agrin, and the monovalent antibodies inhibited the agrin-induced phosphorylation of MuSK. Interestingly, the divalent antibodies from one of the MuSK-MG patients could not induce the MuSK phosphorylation despite their abilities to inhibit the agrininduced AChR clustering, and their modes of action resembled those of the monovalent antibodies. These results demonstrate that the distinct mechanisms between two types of antibodies are involved with the dysfunction of MuSK, and the strong possibility that the functionally monovalent antibodies actually exist in the patients with MuSK-MG. doi:10.1016/j.neures.2011.07.843
e195
Multiple acyl-CoA dehydrogenation deficiency (MADD) is an autosomal recessive disease affecting amino acid, fatty acid, and choline metabolism and is a common genetic defect responsible for lipid-storage myopathy. Most forms of MADD are caused by a deficiency of electron transfer flavoprotein (ETF) or ETF dehydrogenase (ETFDH). However, its molecular feature has not been found uniformly in previous reports of Chinese patients. A large cohort of 56 late-onset MADD patients from 51 unrelated pedigrees in the southern China was recruited to investigate clear correlation between clinical phenotype and molecular genetic basis. All exons of ETFA, ETFB and ETFDH including the intron–exon boundaries, and 5 and 3 untranslated regions were directly sequenced. ETFDH deficiencies affected 94.1% (48/51) of the pedigrees. ETFDH-c.250G>A is the most common mutation, representing a high allelic frequency of 83.3% (80/96). Carrier frequency of c.250G>A is estimated to 1.35% (7/520) in the normal population. A significant reduced expression of ETFDH was identified in muscle of ETFDH-deficient patients. ETFDH deficiency is a major cause of riboflavin-responsive MADD in the southern China and c.250G>A is an important mutation that could be employed as a fast and reliable screening method. Research fund: A key program of scientific research of Fujian Medical University (2009D064), a program for Innovative Research Teams in Science (FMU-RT002), Fuzhou, and a grant from Huashan Hospital for the special professorship of Fudan University, Shanghai (to Wu Z.Y.), China. doi:10.1016/j.neures.2011.07.844
P2-s10 RGMa modulates T cell responses and is involved in autoimmune encephalomyelitis Rieko Muramatsu 1,2 , Takekazu Kubo 3 , Masahiro Mori 4 , Yuka Fujita 1,2 , Satoshi Kuwabara 4 , Toshihide Nakamura 1,2 , Yuki 1,2,3 Yamashita 1
Dep. of Mol. Neurosci., Grad. Sch. of Med., Osaka Univ., Osaka, Japan 2 JSTCREST 3 Dep. of Neurobiol., Grad. Sch. of Med., Chiba University, Chiba, Japan 4 Dep. of Neurol., Grad. Sch. of Med., Chiba University, Chiba, Japan Repulsive guidance molecule-a (RGMa) is a membrane-bound protein that was originally identified as an axon guidance molecule in the development of visual system. Although RGMa is recognized as having a crucial role in the nervous system, the role of RGMa in the immune system is unclear. In this study, we found that RGMa in dendritic cells increase clinical severity of experimental autoimmune encephalomyelitis (EAE) mouse, an animal model of multiple sclerosis (MS). RGMa is expressed in bone marrow-derived dendritic cells (BMDCs) and that CD4+ T cells express receptors for RGMa. We found that RGMa binding to CD4+ T cells led to Rap1 activation and increased adhesion to intracellular adhesion molecule-1 (ICAM-1). Further, treatment with neutralizing antibodies to RGMa prevented mouse myelin oligodendrocyte glycoprotein (MOG)-induced EAE and reduced invasion by inflammatory cells. Adoptive transfer of stimulated BMDCs with RGMa-knockdown or stimulated CD4+ T cells from RGMa-specific antibody-treated EAE mice lead to moderately reduced clinical severity of EAE. CD4+ T cells from mice treated with RGMa-specific antibody had a diminished MOG-specific proliferative response and reduced Interferon-␥ Interleukin-2 (IL-2), IL-4, and IL-17 secretion. T cell response of purified peripheral blood mononuclear cells isolated from individuals with MS also reduced by the treatment with RGMa-specific antibodies. These results suggest that RGMa-specific antibody suppresses the T cell response to antigens; thus, we consider RGMa a promising molecular target for treating MS. doi:10.1016/j.neures.2011.07.845
P2-s11 Prostaglandin I2 -IP signaling promotes migration of oligodendrocyte precursor cell by a mechanism dependent on cAMP Chisato Takahashi 1,2 Yamashita 1,2
, Rieko
Muramatsu 1,2 , Toshihide
1
P2-s09 Molecular analysis of 51 unrelated pedigrees with late-onset multiple acyl-CoA dehydrogenation deficiency (MADD) in the southern China confirmed the most common ETFDH mutation and high carrier frequency of c.250G>A Zhi-Qiang Wang 1 , Xue-Jiao Chen 1 , Shen-Xing Murong 1 , Ning Wang 1 , Zhi-Ying Wu 1,2 1 Department of Neurology and Institute of Neurology, First Affiliated Hospital, Fujian Medical University 2 Department of Neurology and Institute of Neurology, Huashan Hospital, Institutes of Brain Science and State Key Laboratory of Medical Neurobiology, Shanghai Medical College, Fudan University
Dep. of Mol. Neurosci., Grad. Sch. of Med., Osaka Univ., Osaka, Japan 2 Japan Science and Technology Agency, CREST, Tokyo, Japan Oligodendrocytes regulate myelination through axon–oligodendrocyte interaction in the central nervous system (CNS). Formation of myelin sheath around axon enables rapid impulse propagation and protection against axonal damage. Thus, understanding of the mechanisms in CNS myelination and promotion of myelination contribute to recovery of neuronal function in demyelinating disorders, such as multiple sclerosis. Myelin repair after CNS disorders is promoted by migration of oligodendrocyte precursor cells (OPCs); however, the molecular mechanism of OPCs migration remains unclear. Here, we show that prostaglandin I2 (PGI2 ) is a promoter