- Email: [email protected]
Generation of disease-specific induced pluripotent stem cells from Alzheimer's disease patients
Abstracts / Neuroscience Research 68S (2010) e223–e334
own. We previouy reported that two types of AICD were existed. One was soluble and easily degraded form, and the other was membrane associated form. Therfore, we speculate that the accumulation of membrane associated AICD including APP C-terminal fragments may be toxic. In this study, to clear the relationship between the accumulation of AICD and toxic effects, we analzed the metabolism of AICD and the affect of AICD accumulation in cultured cells using proteomic methods. We found that APP C-terminal fragments was degraded in both gamma-secretase pathway and lysosomal pathway. On the proteomic analysis of cultured cells expressing AICDs or cultured cells with gamma-secretase inhibitor treatment, we found that nuclear-related proteins and vesicular trafficking-related proteins reduced in cultured cells expressing membrane-asscociated AICD. We will report and discuss the influence of AICDs accumulation in cultured cells.
e305
cortistatin, corticotropin releasing hormone and 5HT2A receptor may lead to neuronal hypofunction in these regions. On the other hand, male hippocampus and A46 did not present significant age-related differences in any gene sets. These sex differences in brain aging process may result from molecular and cellular deterioration caused by drastic change in hormonal dynamics in aged female monkeys. doi:10.1016/j.neures.2010.07.1354
P2-n14 tert-Butylhydroquinone protects dopaminergic neurons from MPP+ cytotoxicity primarily via normalization of glutathione levels in midbrain slice cultures Yukie Sato , Akinori Hisatsune, Yoichiro Isohama, Hiroshi Katsuki
doi:10.1016/j.neures.2010.07.1352
Dept Chemico-pharmacol Sci, Grad Sch Pharm Sci, Kumamoto Univ, Kumamoto
P2-n12 Generation of disease-specific induced pluripotent stem cells from Alzheimer’s disease patients
Parkinson disease is characterized by selective loss of dopaminergic neurons in the midbrain, which leads to severe motor deficits including tremors and inability to initiate movement. Oxidative stress is implicated in the pathogenic events in this neurodegenerative disorder. Under conditions of increased oxidative stress, a transcription factor NF-E2-related factor (Nrf2) binds to antioxidant response element to induce expression of enzymes related to phase II detoxification and antioxidative defense. Thus, promotion of recruitment of Nrf2 can be a target of neuroprotective therapy that confers resistance to a variety of oxidative stress-related insults on neurons. Here we examined the effect of tert-butylhydroquinone (tBHQ), an activator of Nrf2, against of MPP+ cytotoxicity in rat midbrain slice cultures. Immunohistochemical examinations on tyrosine hydroxylase revealed that tBHQ (5–40 M) inhibited dopaminergic neuron loss induced by 48-h application of 30 M MPP+ , in a concentration-dependent manner. Because Nrf2 activation leads to its translocation to the nucleus, we examined Nrf2 levels in nuclear fractions obtained from midbrain slice homogenates after drug treatment, by western blotting. tBHQ treatment increased Nrf2 accumulation in the nucleus in a concentration-dependent manner, which was accelerated in the presence of MPP+ . We next examined possible involvement of glutathione synthesis and heme oxygenase-1 expression, two major pathways downstream of Nrf2 activation, in the neuroprotective effect of tBHQ. We found that tBHQ significantly increased glutathione content in midbrain slices, and that depletion of glutathione by buthionine sulfoximine abolished the protective effect of tBHQ against MPP+ cytotoxicity. On the other hand, inhibition of heme oxygenase by zinc protoporphyrin IX did not suppress the protective effect of tBHQ. These results suggest that tBHQ protects midbrain dopaminergic neurons via increase in glutathione synthesis.
Naoki Yahata 1 , Harushisa Inoue 1,2 , Shiho Kitaoka 1,2 , Kayoko Tsukita 1,2 , Takayuki Kondo 3 , Naohiro Egawa 1,3 , Isao Asaka 1,2 , Kazutoshi Takahashi 1,2 , Tatsutoshi Nakahata 1 , Shinobu Takahashi 3 , Takashi Kawakatsu 4 , Ryosuke Asada 2,5 , Shinya Yamanaka 1 1
CiRA, iCeMS, Kyoto University 2 JST-CREST 3 Graduate School of Med., Kyoto University 4 Department of Psychiatry, Yamagata University School of Medicine 5 Department of Neuropsychiatry, University of Tsukuba
Alzheimer’s disease (AD) is a neurodegenerative disorder of the central nervous system which causes progressive memory and cognitive decline during middle to late adult life. The cell death mechanisms and therapeutic intervention for AD have been tested by transgenic rodent models. However, these models not fully represent the human condition. Induced pluripotent stem (iPS) cells, which show striking similarities to embryonic stem cells, can now be derived from human adult somatic tissues and have the capacity to be lineage restricted into various neuronal subtypes. Furthermore, establishment of disease-specific human cortical neurons and human cell-based AD model in vitro is crucial for investigation of the disease mechanism and drug discovery. We generated disease-specific iPS cells from patients with AD. Dermal fibroblasts from the patients were transduced with either four or three reprogramming factors, followed by emergence of putative iPS cell colonies after a few weeks. We evaluated their similarity to ES cells by checking the expression of the ES cell- associated antigens. Next, we examined the pluripotency of iPS cells by teratoma formation and embryoid body (EB) differentiation in vitro. Disease-specific iPS cells from AD patients possess properties of ES cells with patient-specific genetic information and may contribute to explore the pathogenesis of AD. doi:10.1016/j.neures.2010.07.1353
P2-n13 Microarray profiling of gene expression in the aging monkey brain Sayuri
Higaki 1
, Akira
Sato 2 ,
Toshio
Kojima 2,3 ,
Takao
Oishi 1
1
Sect Systems Neurosci, Primate Res Inst, Kyoto Univ, Inuyama Computational Systems Biol Res Group, RIKEN, Yokohama 3 Res Equipment Center, Hamamatsu Univ Sch of Med, Hamamatsu
2
Aging of the primate proceeds slowly and is characterized by prominent senescence, i.e., prolonged survival after reproductive years. To further our understanding of the characteristic aging process of the primate brain, we conducted DNA microarray analysis of Brodmann’s area 46 (A46) and hippocampus in aged monkeys, because these areas are thought to be important for the process of learning and memory. Monkeys (n = 24) were assigned into 4 groups based on age (young; mean age 10.0 and aged; mean age 26.3) and sex (female and male). RNA isolated from 48 brain tissues was applied to the 4 × 44K Rhesus Monkey Oligo Microarrays (Agilent). For 1205 genes expressed differentially more than 1.5 fold between young and aged monkey hippocampus or A46, we employed GSEA (Gene Set Enrichment Analysis) to evaluate whether pre-defined set of genes shows statistically significant, concordant differences. In aged female hippocampus but not male hippocampus, homeostasis-related gene sets were significantly upregulated (FDR q-value < 0.25). The hyper-expression of genes encoding chemokine and complement proteins engaged in immunoregulatory and anti-inflammatory activity implies age-related inflammation or disease progression. Moreover, in aged female hippocampus and A46, gene set involved in neurological system process was significantly downregulated (FDR q-value < 0.25). The decreased expression of genes encoding neuropeptide Y, somatostatin,
doi:10.1016/j.neures.2010.07.1355
P2-n17 Human DJ-1-specific transcriptional activation of the tyrosine hydroxylase gene Shizuma Ishikawa 1 , Takahiro Taira 2 , Kazuko Takahashi-Niki 3 , Takeshi Niki 1 , Hiroyoshi Ariga 3 , Sanae M.M. Iguchi-Ariga 1 1
Grad. Agr. Hokkaido University 2 Grad. Med. Yamanashi. University 3 Grad. Pharm. Sci. Hokkaido University
Loss-of-functional mutation in the DJ-1 gene causes a subset of familial Parkinson disease. The mechanism underlying DJ-1-related selective vulnerability in the dopaminergic pathway is, however, not resolved. DJ-1 has multiple functions, including transcriptional regulation, and one of the transcriptional target genes for DJ-1 is the tyrosine hydroxylase (TH) gene whose product is a key enzyme for dopamine biosynthesis. It has been reported that DJ-1 is a neuroprotective transcriptional co-activator that sequesters a transcriptional co-repressor pyrimidine tract-binding protein associated splicing factor (PSF) from the TH gene promoter. In this study, we found that knockdown of human DJ-1 by small interference RNA in human dopaminergic cell lines attenuated the TH gene expression and L-DOPA production but that knockdown or knockout of mouse DJ-1 in mouse cell lines or in DJ-1 knockout mice did not affect such expression and activity of TH. Reporter assays using the human TH gene promoter linked to the luciferase gene showed that stimulation of TH promoter activity was observed in human but not mouse cells transfected with DJ-1. Although human and mouse DJ-1 were associated either with human or with mouse PSF, TH promoter activity inhibited by PSF was restored by human DJ-1 but not mouse DJ-1. Chromatin immunoprecipitation assays revealed that the complex of PSF with DJ-1 bound to the human but not mouse TH gene promoter. These results suggest a novel
own. We previouy reported that two types of AICD were existed. One was soluble and easily degraded form, and the other was membrane associated form. Therfore, we speculate that the accumulation of membrane associated AICD including APP C-terminal fragments may be toxic. In this study, to clear the relationship between the accumulation of AICD and toxic effects, we analzed the metabolism of AICD and the affect of AICD accumulation in cultured cells using proteomic methods. We found that APP C-terminal fragments was degraded in both gamma-secretase pathway and lysosomal pathway. On the proteomic analysis of cultured cells expressing AICDs or cultured cells with gamma-secretase inhibitor treatment, we found that nuclear-related proteins and vesicular trafficking-related proteins reduced in cultured cells expressing membrane-asscociated AICD. We will report and discuss the influence of AICDs accumulation in cultured cells.
e305
cortistatin, corticotropin releasing hormone and 5HT2A receptor may lead to neuronal hypofunction in these regions. On the other hand, male hippocampus and A46 did not present significant age-related differences in any gene sets. These sex differences in brain aging process may result from molecular and cellular deterioration caused by drastic change in hormonal dynamics in aged female monkeys. doi:10.1016/j.neures.2010.07.1354
P2-n14 tert-Butylhydroquinone protects dopaminergic neurons from MPP+ cytotoxicity primarily via normalization of glutathione levels in midbrain slice cultures Yukie Sato , Akinori Hisatsune, Yoichiro Isohama, Hiroshi Katsuki
doi:10.1016/j.neures.2010.07.1352
Dept Chemico-pharmacol Sci, Grad Sch Pharm Sci, Kumamoto Univ, Kumamoto
P2-n12 Generation of disease-specific induced pluripotent stem cells from Alzheimer’s disease patients
Parkinson disease is characterized by selective loss of dopaminergic neurons in the midbrain, which leads to severe motor deficits including tremors and inability to initiate movement. Oxidative stress is implicated in the pathogenic events in this neurodegenerative disorder. Under conditions of increased oxidative stress, a transcription factor NF-E2-related factor (Nrf2) binds to antioxidant response element to induce expression of enzymes related to phase II detoxification and antioxidative defense. Thus, promotion of recruitment of Nrf2 can be a target of neuroprotective therapy that confers resistance to a variety of oxidative stress-related insults on neurons. Here we examined the effect of tert-butylhydroquinone (tBHQ), an activator of Nrf2, against of MPP+ cytotoxicity in rat midbrain slice cultures. Immunohistochemical examinations on tyrosine hydroxylase revealed that tBHQ (5–40 M) inhibited dopaminergic neuron loss induced by 48-h application of 30 M MPP+ , in a concentration-dependent manner. Because Nrf2 activation leads to its translocation to the nucleus, we examined Nrf2 levels in nuclear fractions obtained from midbrain slice homogenates after drug treatment, by western blotting. tBHQ treatment increased Nrf2 accumulation in the nucleus in a concentration-dependent manner, which was accelerated in the presence of MPP+ . We next examined possible involvement of glutathione synthesis and heme oxygenase-1 expression, two major pathways downstream of Nrf2 activation, in the neuroprotective effect of tBHQ. We found that tBHQ significantly increased glutathione content in midbrain slices, and that depletion of glutathione by buthionine sulfoximine abolished the protective effect of tBHQ against MPP+ cytotoxicity. On the other hand, inhibition of heme oxygenase by zinc protoporphyrin IX did not suppress the protective effect of tBHQ. These results suggest that tBHQ protects midbrain dopaminergic neurons via increase in glutathione synthesis.
Naoki Yahata 1 , Harushisa Inoue 1,2 , Shiho Kitaoka 1,2 , Kayoko Tsukita 1,2 , Takayuki Kondo 3 , Naohiro Egawa 1,3 , Isao Asaka 1,2 , Kazutoshi Takahashi 1,2 , Tatsutoshi Nakahata 1 , Shinobu Takahashi 3 , Takashi Kawakatsu 4 , Ryosuke Asada 2,5 , Shinya Yamanaka 1 1
CiRA, iCeMS, Kyoto University 2 JST-CREST 3 Graduate School of Med., Kyoto University 4 Department of Psychiatry, Yamagata University School of Medicine 5 Department of Neuropsychiatry, University of Tsukuba
Alzheimer’s disease (AD) is a neurodegenerative disorder of the central nervous system which causes progressive memory and cognitive decline during middle to late adult life. The cell death mechanisms and therapeutic intervention for AD have been tested by transgenic rodent models. However, these models not fully represent the human condition. Induced pluripotent stem (iPS) cells, which show striking similarities to embryonic stem cells, can now be derived from human adult somatic tissues and have the capacity to be lineage restricted into various neuronal subtypes. Furthermore, establishment of disease-specific human cortical neurons and human cell-based AD model in vitro is crucial for investigation of the disease mechanism and drug discovery. We generated disease-specific iPS cells from patients with AD. Dermal fibroblasts from the patients were transduced with either four or three reprogramming factors, followed by emergence of putative iPS cell colonies after a few weeks. We evaluated their similarity to ES cells by checking the expression of the ES cell- associated antigens. Next, we examined the pluripotency of iPS cells by teratoma formation and embryoid body (EB) differentiation in vitro. Disease-specific iPS cells from AD patients possess properties of ES cells with patient-specific genetic information and may contribute to explore the pathogenesis of AD. doi:10.1016/j.neures.2010.07.1353
P2-n13 Microarray profiling of gene expression in the aging monkey brain Sayuri
Higaki 1
, Akira
Sato 2 ,
Toshio
Kojima 2,3 ,
Takao
Oishi 1
1
Sect Systems Neurosci, Primate Res Inst, Kyoto Univ, Inuyama Computational Systems Biol Res Group, RIKEN, Yokohama 3 Res Equipment Center, Hamamatsu Univ Sch of Med, Hamamatsu
2
Aging of the primate proceeds slowly and is characterized by prominent senescence, i.e., prolonged survival after reproductive years. To further our understanding of the characteristic aging process of the primate brain, we conducted DNA microarray analysis of Brodmann’s area 46 (A46) and hippocampus in aged monkeys, because these areas are thought to be important for the process of learning and memory. Monkeys (n = 24) were assigned into 4 groups based on age (young; mean age 10.0 and aged; mean age 26.3) and sex (female and male). RNA isolated from 48 brain tissues was applied to the 4 × 44K Rhesus Monkey Oligo Microarrays (Agilent). For 1205 genes expressed differentially more than 1.5 fold between young and aged monkey hippocampus or A46, we employed GSEA (Gene Set Enrichment Analysis) to evaluate whether pre-defined set of genes shows statistically significant, concordant differences. In aged female hippocampus but not male hippocampus, homeostasis-related gene sets were significantly upregulated (FDR q-value < 0.25). The hyper-expression of genes encoding chemokine and complement proteins engaged in immunoregulatory and anti-inflammatory activity implies age-related inflammation or disease progression. Moreover, in aged female hippocampus and A46, gene set involved in neurological system process was significantly downregulated (FDR q-value < 0.25). The decreased expression of genes encoding neuropeptide Y, somatostatin,
doi:10.1016/j.neures.2010.07.1355
P2-n17 Human DJ-1-specific transcriptional activation of the tyrosine hydroxylase gene Shizuma Ishikawa 1 , Takahiro Taira 2 , Kazuko Takahashi-Niki 3 , Takeshi Niki 1 , Hiroyoshi Ariga 3 , Sanae M.M. Iguchi-Ariga 1 1
Grad. Agr. Hokkaido University 2 Grad. Med. Yamanashi. University 3 Grad. Pharm. Sci. Hokkaido University
Loss-of-functional mutation in the DJ-1 gene causes a subset of familial Parkinson disease. The mechanism underlying DJ-1-related selective vulnerability in the dopaminergic pathway is, however, not resolved. DJ-1 has multiple functions, including transcriptional regulation, and one of the transcriptional target genes for DJ-1 is the tyrosine hydroxylase (TH) gene whose product is a key enzyme for dopamine biosynthesis. It has been reported that DJ-1 is a neuroprotective transcriptional co-activator that sequesters a transcriptional co-repressor pyrimidine tract-binding protein associated splicing factor (PSF) from the TH gene promoter. In this study, we found that knockdown of human DJ-1 by small interference RNA in human dopaminergic cell lines attenuated the TH gene expression and L-DOPA production but that knockdown or knockout of mouse DJ-1 in mouse cell lines or in DJ-1 knockout mice did not affect such expression and activity of TH. Reporter assays using the human TH gene promoter linked to the luciferase gene showed that stimulation of TH promoter activity was observed in human but not mouse cells transfected with DJ-1. Although human and mouse DJ-1 were associated either with human or with mouse PSF, TH promoter activity inhibited by PSF was restored by human DJ-1 but not mouse DJ-1. Chromatin immunoprecipitation assays revealed that the complex of PSF with DJ-1 bound to the human but not mouse TH gene promoter. These results suggest a novel