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The role of macroautophagy in the pathogenesis of neuronal ceroid lipofuscinoses due to cathepsin D deficiency
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Abstracts / Neuroscience Research 71S (2011) e108–e415
gen receptor (AR) gene. The pathogenic AR with expanded polyglutamine (polyQ) accumulates in the nucleus, and inhibits transcription by interfering with transcriptional factors and co-activators. Leuprorelin suppressed the accumulation of pathogenic androgen receptors in a phase 2 trial in SBMA patients, and is expected to inhibit or slow down the pathogenic processes of neuronal degeneration. However, even when leuprorelin stops the disease progression, motor improvements are not evident. Thus, to achieve a global improvement in personal well-being, not only disease-modifying therapies, but also symptom-relief therapies, such as l-DOPA for Parkinson’s disease should be developed. For this purpose, valid complementary therapeutic interventions for SBMA treatment should be extensively investigated. We show that overexpression of hepatocyte growth factor (HGF) in the nervous system attenuates motor impairment and prolongs the life span of the SBMA transgenic mice. Furthermore, overexpression of HGF exerts additive therapeutic effects in the SBMA transgenic mice. These observations suggest that HGF is a promising symptom-relief therapeutic candidate for polyQ-mediated neurodegenerative diseases including SBMA as a combination therapy with an anti-androgen therapy. Research fund: KAKENHI (21659220) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. doi:10.1016/j.neures.2011.07.837
P2-s03 Identification of a novel binding partner for FUS/TLS Sakiko Fujii 1 , Hideaki Miyachi 1 , Syunsuke Koga 2 , Keiko Kitajyo 1 , Atsushi Yamaguchi 1 1
Div. of Neurobio., Grad. Sch. of Med., Chiba Univ., Chiba, Japan Neurology., Grad. Sch. of Med., Chiba Univ., Chiba, Japan
2
Div. of
The fused in sarcoma/translated in sarcoma (FUS/TLS) mutation has been identified as a cause of familial amyotrophic lateral sclerosis (ALS). Postmortem analysis with FUS/TLS mutations found abnormal FUS/TLS cytoplasmic inclusions in neurons and glial cells. The FUS/TLS gene encodes a 526 amino acid protein characterized by an N-terminal QGSY region, an RRM (RNA recognition motif), and C-terminal multiple RGG repeats, suggesting its roles in general mRNA and miRNA processing. In order to identify the binding partners, we performed a yeast two-hybrid screening on human fetal brain cDNA library. One possible interacting partner, ProteinX, is a major methyltransferase in mammalian cells. In this presentation, we would show the preliminary results on the functional interplay between FUS/TLS and Protein X. doi:10.1016/j.neures.2011.07.838
P2-s04 Induction of transglutaminase in ALS model mice Oono 1
Ido 2 ,
Matsumoto 3 ,
Miki , Akemi Ayako Taniguchi 3 , Ryosuke Takahashi 1 , Makoto Urushitani 2
Naoyuki
1
Dep. of Neurology, Grad. Sch. of Med, Kyoto Univ., Kyoto, Japan 2 Unit for Neurobiology and Therapeutics, Molecular Neuroscience Research Center (MNRC), Shiga University of Medical Science, Setatsukinowa-cho, Otsu, Japan 3 Department of Disease Glycomics, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Japan Introduction: Accumulating evidence indicates that amyotrophic lateral sclerosis (ALS)-linked mutant SOD1 (mSOD1) distributes in the ER–Golgi system and is secreted. Extracellular SOD1 mutant-activates microglia and causes the motoneuron death. We previously reported the effect of vaccination against mSOD1 transgenic mice with mutant or WT SOD1 vaccination. However, the effect is not satisfactory and it is important to identify more pathogenic target, such as oligomer. Here we focus on transglutaminase2 (TGM2) as a candidate generator of mSOD1 oligomer, and examined the TGM2 expression profile in ALS model mice. Method: E. coli-purified recombinant SOD1 proteins (WT, G93A) were incubated with human TGM2 recombinant protein and the oligomerization of SOD1 was investigated by Western blotting. To examine the detergent solubility of SOD1 oligomer, Neuro2a cells were co-transfected with three types of TGM2 (WT, C277S; inactive mutant, R580A; mutant of exhibiting activity at much lower calcium concentration) together with SOD1 (G93A, WT). After the separation into detergent-soluble and -insoluble fractions, each fraction of cell lysates was analyzed by Western blotting. The expression of TGM2 was investigated by the real-time PCR analysis in the hypoglossal nucleus of non-Tg C57Bl/6 mice with the nerve ligation, or of the presymptomatic G93A SOD1 Tg mice. Results and conclusions: Recombinant human TGM2 oligomerized mutant and apo-WT SOD1, but not WT holo SOD1. In transfected Neuro2a cells, the high molecular SOD1 aggregates of the mutant, but not WT, were detected only
in the detergent-insoluble fraction in the cells expressing TGM2 of WT and R580A, but not of TGM2 of C277S. TGM2 was significantly induced by the axonal ligation both in non-Tg and G93A Tg mice. Future plans: The generation of large amount of SOD1 oligomers and the modification of the adjuvant system to induce the protective acquired immunity is underway. doi:10.1016/j.neures.2011.07.839
P2-s05 CRAG protects neuronal cells against cytotoxicity of expanded polyglutamine protein partially via c-fosdependent AP-1 activation Shun Nagashima , Toshifumi Fukuda, Ayaka Iwase, Kouhei Miura, Ryoko Inatome, Shigeru Yanagi Lab. of Mol. Biochem., Sch. of Life Sci., Tokyo Univ. of Pharm. and Life Sci. Tokyo, Japan Polyglutamine diseases are neurodegenerative disorders whose treatment is not established. We have previously shown that CRAG, a short splicing variant of centaurin-␥3/AGAP3, facilitated the degradation of expanded polyglutamine protein (polyQ) via the nuclear ubiquitin-proteasome pathway. Taking advantage of this feature, lentivirus-mediated CRAG expression in Purkinje cells of model mice expressing polyQ cleared polyQ aggregates and rescued from ataxia. However, the molecular basis of CRAG function in cell survival against polyQ remains unclear. Here we report that CRAG, but not centaurin-␥3, induces transcriptional activation of c-fos-dependent activator protein-1 (AP-1). Mutation analysis indicated that the nuclear localization signal and both N- and C-terminal regions of CRAG were critical for the c-fos activation. CRAG knockdown by siRNA or expression of CRAG dominant negative mutant significantly attenuated the c-fos activation triggered by either polyQ or proteasome inhibitor MG132. Furthermore, c-fos expression partially rescued from the enhanced cytotoxicity by CRAG knockdown in polyQ-expressing or MG132-treated cells. Thus, CRAG strongly enhances cell survival signal against accumulation of unfolded proteins including polyQ through not only the proteasome activation but also activation of c-fosdependent AP-1. Our findings further extend the usefulness of targeted delivery of CRAG as a gene therapy for polyglutamine diseases. doi:10.1016/j.neures.2011.07.840
P2-s06 The role of macroautophagy in the pathogenesis of neuronal ceroid lipofuscinoses due to cathepsin D deficiency Masato Koike , Yasuo Uchiyama Department of Cell Biology and Neurosciences, Juntedo University Faculty of Medicine, Tokyo, Japan Neurons of cathepsin D-deficient (CD−/−) mice, a murine model for neuronal ceroid lipofuscinoses (NCLs), possess abundant autophagosomes and granular osmiophilic deposits (GRODs), which are morphological hallmarks of NCLs. Moreover, GRODs are often found in nascent autophagosomes, indicating that the GROD is a potent inducer of autophagy in neuronal cells. In fact, using immunohistochemistry for LC3, ubiquitin, and p62 that can interact with both ubiquitin and LC3, we further confirmed that ubiquitin and p62 are co-localized on the membrane of GRODs, while these three molecules are present in the same granules of the neurons. In the double knockout mice lacking cathepsin D and Atg7 specifically in CNS tissue, although GRODs still existed in the neuronal perikarya, suggesting that GRODs are generated via unknown intracellular routes to lysosomes other than autophagy. Moreover, instead of being enwrapped by smooth double membranes, GRODs were frequently surrounded by accumulated profiles of rough ER, while such structures gathered in certain perikaryal areas, forming cytosolic aggregates. On the other hand, no clear-cut difference was detected in the neuropathological phenotypes between CD−/− mice and double knockout mice lacking cathepsin D and p62. In fact we found that Nbr1, which has been shown to interact both ubiquitin and p62, also localize on the surface of GRODs together with p62 in CD−/− mice. These data indicate that ubiquitin and p62 and/or Nbr1 on the membrane of GRODs may serve as a signal of autophagy that contributes to the pathogenesis of NCLs. Research fund: KAKENHI (21790191, 21025030) and The Science Research Promotion Fund. doi:10.1016/j.neures.2011.07.841
Abstracts / Neuroscience Research 71S (2011) e108–e415
gen receptor (AR) gene. The pathogenic AR with expanded polyglutamine (polyQ) accumulates in the nucleus, and inhibits transcription by interfering with transcriptional factors and co-activators. Leuprorelin suppressed the accumulation of pathogenic androgen receptors in a phase 2 trial in SBMA patients, and is expected to inhibit or slow down the pathogenic processes of neuronal degeneration. However, even when leuprorelin stops the disease progression, motor improvements are not evident. Thus, to achieve a global improvement in personal well-being, not only disease-modifying therapies, but also symptom-relief therapies, such as l-DOPA for Parkinson’s disease should be developed. For this purpose, valid complementary therapeutic interventions for SBMA treatment should be extensively investigated. We show that overexpression of hepatocyte growth factor (HGF) in the nervous system attenuates motor impairment and prolongs the life span of the SBMA transgenic mice. Furthermore, overexpression of HGF exerts additive therapeutic effects in the SBMA transgenic mice. These observations suggest that HGF is a promising symptom-relief therapeutic candidate for polyQ-mediated neurodegenerative diseases including SBMA as a combination therapy with an anti-androgen therapy. Research fund: KAKENHI (21659220) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. doi:10.1016/j.neures.2011.07.837
P2-s03 Identification of a novel binding partner for FUS/TLS Sakiko Fujii 1 , Hideaki Miyachi 1 , Syunsuke Koga 2 , Keiko Kitajyo 1 , Atsushi Yamaguchi 1 1
Div. of Neurobio., Grad. Sch. of Med., Chiba Univ., Chiba, Japan Neurology., Grad. Sch. of Med., Chiba Univ., Chiba, Japan
2
Div. of
The fused in sarcoma/translated in sarcoma (FUS/TLS) mutation has been identified as a cause of familial amyotrophic lateral sclerosis (ALS). Postmortem analysis with FUS/TLS mutations found abnormal FUS/TLS cytoplasmic inclusions in neurons and glial cells. The FUS/TLS gene encodes a 526 amino acid protein characterized by an N-terminal QGSY region, an RRM (RNA recognition motif), and C-terminal multiple RGG repeats, suggesting its roles in general mRNA and miRNA processing. In order to identify the binding partners, we performed a yeast two-hybrid screening on human fetal brain cDNA library. One possible interacting partner, ProteinX, is a major methyltransferase in mammalian cells. In this presentation, we would show the preliminary results on the functional interplay between FUS/TLS and Protein X. doi:10.1016/j.neures.2011.07.838
P2-s04 Induction of transglutaminase in ALS model mice Oono 1
Ido 2 ,
Matsumoto 3 ,
Miki , Akemi Ayako Taniguchi 3 , Ryosuke Takahashi 1 , Makoto Urushitani 2
Naoyuki
1
Dep. of Neurology, Grad. Sch. of Med, Kyoto Univ., Kyoto, Japan 2 Unit for Neurobiology and Therapeutics, Molecular Neuroscience Research Center (MNRC), Shiga University of Medical Science, Setatsukinowa-cho, Otsu, Japan 3 Department of Disease Glycomics, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Japan Introduction: Accumulating evidence indicates that amyotrophic lateral sclerosis (ALS)-linked mutant SOD1 (mSOD1) distributes in the ER–Golgi system and is secreted. Extracellular SOD1 mutant-activates microglia and causes the motoneuron death. We previously reported the effect of vaccination against mSOD1 transgenic mice with mutant or WT SOD1 vaccination. However, the effect is not satisfactory and it is important to identify more pathogenic target, such as oligomer. Here we focus on transglutaminase2 (TGM2) as a candidate generator of mSOD1 oligomer, and examined the TGM2 expression profile in ALS model mice. Method: E. coli-purified recombinant SOD1 proteins (WT, G93A) were incubated with human TGM2 recombinant protein and the oligomerization of SOD1 was investigated by Western blotting. To examine the detergent solubility of SOD1 oligomer, Neuro2a cells were co-transfected with three types of TGM2 (WT, C277S; inactive mutant, R580A; mutant of exhibiting activity at much lower calcium concentration) together with SOD1 (G93A, WT). After the separation into detergent-soluble and -insoluble fractions, each fraction of cell lysates was analyzed by Western blotting. The expression of TGM2 was investigated by the real-time PCR analysis in the hypoglossal nucleus of non-Tg C57Bl/6 mice with the nerve ligation, or of the presymptomatic G93A SOD1 Tg mice. Results and conclusions: Recombinant human TGM2 oligomerized mutant and apo-WT SOD1, but not WT holo SOD1. In transfected Neuro2a cells, the high molecular SOD1 aggregates of the mutant, but not WT, were detected only
in the detergent-insoluble fraction in the cells expressing TGM2 of WT and R580A, but not of TGM2 of C277S. TGM2 was significantly induced by the axonal ligation both in non-Tg and G93A Tg mice. Future plans: The generation of large amount of SOD1 oligomers and the modification of the adjuvant system to induce the protective acquired immunity is underway. doi:10.1016/j.neures.2011.07.839
P2-s05 CRAG protects neuronal cells against cytotoxicity of expanded polyglutamine protein partially via c-fosdependent AP-1 activation Shun Nagashima , Toshifumi Fukuda, Ayaka Iwase, Kouhei Miura, Ryoko Inatome, Shigeru Yanagi Lab. of Mol. Biochem., Sch. of Life Sci., Tokyo Univ. of Pharm. and Life Sci. Tokyo, Japan Polyglutamine diseases are neurodegenerative disorders whose treatment is not established. We have previously shown that CRAG, a short splicing variant of centaurin-␥3/AGAP3, facilitated the degradation of expanded polyglutamine protein (polyQ) via the nuclear ubiquitin-proteasome pathway. Taking advantage of this feature, lentivirus-mediated CRAG expression in Purkinje cells of model mice expressing polyQ cleared polyQ aggregates and rescued from ataxia. However, the molecular basis of CRAG function in cell survival against polyQ remains unclear. Here we report that CRAG, but not centaurin-␥3, induces transcriptional activation of c-fos-dependent activator protein-1 (AP-1). Mutation analysis indicated that the nuclear localization signal and both N- and C-terminal regions of CRAG were critical for the c-fos activation. CRAG knockdown by siRNA or expression of CRAG dominant negative mutant significantly attenuated the c-fos activation triggered by either polyQ or proteasome inhibitor MG132. Furthermore, c-fos expression partially rescued from the enhanced cytotoxicity by CRAG knockdown in polyQ-expressing or MG132-treated cells. Thus, CRAG strongly enhances cell survival signal against accumulation of unfolded proteins including polyQ through not only the proteasome activation but also activation of c-fosdependent AP-1. Our findings further extend the usefulness of targeted delivery of CRAG as a gene therapy for polyglutamine diseases. doi:10.1016/j.neures.2011.07.840
P2-s06 The role of macroautophagy in the pathogenesis of neuronal ceroid lipofuscinoses due to cathepsin D deficiency Masato Koike , Yasuo Uchiyama Department of Cell Biology and Neurosciences, Juntedo University Faculty of Medicine, Tokyo, Japan Neurons of cathepsin D-deficient (CD−/−) mice, a murine model for neuronal ceroid lipofuscinoses (NCLs), possess abundant autophagosomes and granular osmiophilic deposits (GRODs), which are morphological hallmarks of NCLs. Moreover, GRODs are often found in nascent autophagosomes, indicating that the GROD is a potent inducer of autophagy in neuronal cells. In fact, using immunohistochemistry for LC3, ubiquitin, and p62 that can interact with both ubiquitin and LC3, we further confirmed that ubiquitin and p62 are co-localized on the membrane of GRODs, while these three molecules are present in the same granules of the neurons. In the double knockout mice lacking cathepsin D and Atg7 specifically in CNS tissue, although GRODs still existed in the neuronal perikarya, suggesting that GRODs are generated via unknown intracellular routes to lysosomes other than autophagy. Moreover, instead of being enwrapped by smooth double membranes, GRODs were frequently surrounded by accumulated profiles of rough ER, while such structures gathered in certain perikaryal areas, forming cytosolic aggregates. On the other hand, no clear-cut difference was detected in the neuropathological phenotypes between CD−/− mice and double knockout mice lacking cathepsin D and p62. In fact we found that Nbr1, which has been shown to interact both ubiquitin and p62, also localize on the surface of GRODs together with p62 in CD−/− mice. These data indicate that ubiquitin and p62 and/or Nbr1 on the membrane of GRODs may serve as a signal of autophagy that contributes to the pathogenesis of NCLs. Research fund: KAKENHI (21790191, 21025030) and The Science Research Promotion Fund. doi:10.1016/j.neures.2011.07.841