- Email: [email protected]
The investigation into the degradation of the Notch3 protein in the stable cell lines expressing the wild and mutant Notch3
Abstracts / Neuroscience Research 68S (2010) e223–e334
P2-o04 The investigation into the degradation of the Notch3 protein in the stable cell lines expressing the wild and mutant Notch3 Akiko Hosomi 1 , Toshiki Mizuno 1 , Yoshihisa Watanabe 2 , Masaki Tanaka 2 , Masanori Nakagawa 1 1
Department of Neurology, Kyoto Prefectural University of Medicine, Kyoto Department of Cell Biology Research Institute for Neurological Disease and Geriatrics 2
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) is caused by a point mutation that encodes the extracellular domain of NOTCH3 (ECD-N3). It has not been known how CADASIL mutated N3 lead to the degeneration of vascular smooth muscle cells. Previous reports show that the ECD-N3 mutant protein abnormally accumulated in the arteries from the CADASIL patients. We assume that the proteolysis of ECD-N3 or the N3-processing pathway is impaired by the mutations. To address this hypothesis, we generated stable cell lines expressing the mutant (C185R) or the wild type N3 using the Flp-In T-Rex system. A single copy of these vectors was integrated at the FRT site in Flp-In T-Rex 293 host cell, and the expression of N3 was regulated by the addition of tetracycline. To determine the half-life of each N3 protein, these stable cells were grown in the absence of tetracycline for 0–7days. The half-lives of full length, ECD or intracellular domain N3 proteins were measured by western blotting analysis. There was no difference between the degradation rates of wild–type and C185R-mutant N3. However, immunocytochemical analysis of these stable cells identified that both N3 protein, in part, accumulated intracellularly. We then measured the degradation of N3 protein on cell surface by biotinylation experiment. We will discuss the relationship between CADASIL mutation and N3 protein metabolism. doi:10.1016/j.neures.2010.07.1371
P2-o05 Protective effect of hesperidin and naringin against 3 nitropropionic acid induced Huntingtons like symptoms in rats possible role of nitric oxide Puneet Kumar , Anil Kumar Pharmacology, Panjab University 3-nitropropionic acid (3-NP) is a well known experimental model to study Huntingtons disease (HD) and associated neuropsychiatric problems. Present study has been designed to explore the protective effects of hesperidin, naringin, and their nitric oxide mechanism (if any) against 3-nitropropionic acid induced neurotoxicity in rats. Systemic 3-nitropropionic acid (10 mg/kg) treatment for 14 days in rats significantly induced HD like symptoms in rats as indicated by reduced locomotor activity, body weight, grip strength, oxidative defense and mitochondrial complex enzymes (complex I, II, and IV) activities in striatum. Naringin and hesperidin pretreatment significantly attenuated behavioural alterations, oxidative stress and mitochondrial enzyme complex dysfunction in 3-NP treated group. l-arginine (50 mg/kg) pretreatment with lower dose of hesperidin (50 mg/kg) and naringin (50 mg/kg) significantly attenuated their protective effect respectively. Whereas l-NAME (10 mg/kg), a non selective NOS inhibitor pretreatment with hesperidin (50 mg/kg) and naringin (50 mg/kg) significantly potentiated their protective effect which was significant as compared to their effect per se. Study highlights the therapeutic potential of hesperidin and naringin against Huntingtons like conditions and further indicates that these drugs might act through nitric oxide mechanism. doi:10.1016/j.neures.2010.07.1372
P2-o06 Melanin-concentrating hormone neurons, rather than orexin neurons, are vulnerable to quinolinate cytotoxicity in vivo Kanae Obukuro , Moeko Takigawa, Akinori Hisatsune, Yoichiro Isohama, Hiroshi Katsuki Department of Chemico-Pharmacological Sciences, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto Orexins are neuropeptides produced in the lateral hypothalamus (LH) that play an important role in regulation of sleep–wake cycle. Several studies indicate that selective loss of orexin neurons causes narcolepsy, but the mechanisms of this pathological change remain unclear. A previous study showed that quinolinic acid (QA), acting as an endogenous agonist at N-methyl-d-aspartate (NMDA) receptors, caused selective loss of orexin neurons in rat hypothalamic slice culture, with melanin-concentrating hormone (MCH) neurons preserved. Here we examined QA cytotoxicity in vivo.
e309
QA was injected into LH of adult C57BL/6 mice, and frozen brain sections were obtained three days later for double immunofluorescence staining of orexin and MCH. The number of positive cells of all sections of QA-injected side was compared with control side. Unexpectedly, injection of QA (60 and 120 nmol) caused a selective and dose-dependent decrease of the number of MCH neurons while having no significant effect on the number of orexin neurons. This toxicity was attenuated by 10 nmol MK-801, an NMDA receptor antagonist. Next we examined whether QA cytotoxicity could be affected when GABAergic inhibitory inputs were shut off. Injection of QA (60 nmol) with GABAA receptor antagonists such as bicuculline (1 nmol) and picrotoxin (2 nmol) again caused selective loss of MCH neurons as compared to orexin neurons. A significant decrease in the number of orexin neurons was induced when QA injection was performed in the dark phase of diurnal cycle (14.8% and 21.8% reduction by 60 and 120 nmol QA respectively), but the degree of the decrease was still lower than that in the number of MCH neurons (40.8% and 57.4% reduction by 60 and 120 nmol QA respectively). Finally, selective loss of MCH neurons by injection of QA (60 nmol) was also observed in Wistar rats at 3–4 weeks of age. These results do not support the hypothesis that acute excitotoxicity is responsible for the pathogenesis of narcolepsy. doi:10.1016/j.neures.2010.07.1373
P2-o07 The localization of the C terminal fragment of ␣1A calcium channel influences the CREB target transcription Makoto Takahashi , Kinya Ishikawa, Tarou Ishiguro, Masato Obayashi, Hidehiro Mizuisawa Dept of Neurology and Neurological Science, Tokyo Medical and Dental University, Tokyo Background: Spinocerebellar ataxia type6 (SCA6) is considered one of nine polyglutamine diseases, which is caused by the polyglutamine(polyQ) repeat expansion, and the cause of SCA6 is the polyQ repeat expansion of the Carboxyl(C) terminal of ␣1A voltage-gated calcium channel(Cav2.1), but the basic mechanism for neurodegeneration has been unknown. The C terminal fragment (CTF) of Cav2.1,which may be cleaved from the full length Cav2.1, basically locates in the cytoplasm, but recent reports found the possibility that CTF is tend to locate in the nucleus, when the polyQ length of CTF are expanded. Object: To examine whether the location of CTF of Cav2.1 influences the transcription machinery and pathomechanism of SCA6. Method: At first, we made the various polyQ length of CTF vectors, with nuclear export signal (NES) or nuclear localization signal (NLS). With transient expression cell models, we checked the influence of CTF for CREB target transcripition with the use of CRE-luciferase assay and GAL4-CREB-luciferase assay. Result: When the CTF of Cav2.1 locates in the nucleus, CREB target transcription are suppressed and when they locate in the cytoplasm, CREB target transcription are increased. As the polyQ length expands, the transcription activity is gradually increased, but when the polyQ length is too long, the transcription is suppressed. Conclusion: The localization and polyQ length of CTF of Cav2.1 may influence the CREB target transcription. doi:10.1016/j.neures.2010.07.1374
P2-o08 The therapeutic agent NDDPX08 promotes sustainable motor performance and extends a post-onset survival interval in an ALS mouse model Kazunori Tanaka 1 , Takuya Kanno 2 , Yoshiko Yanagisawa 2 , Masashi Aoki 3 , Shinji Hadano 2 , Yasuto Itoyama 3 , Mieko Ogino 4 , Yasuo Iwasaki 5 , Fumihito Yoshii 6 , Joh-E. Ikeda 1,2 1
Neugen Pharma Inc. Kanagawa 2 Dept of Mol Life Sci, Tokai Univ Scl of Med, Kanagawa 3 Dept of Neurol, Tohoku Univ Grad Sch of Med, Miyagi 4 Dept of Neurol, Kitasato Univ East Hospital, Kanagawa 5 Dept of Neurol, Toho Univ Omori Hospital, Tokyo 6 Dept of Neurol, Tokai Univ Sch of Med, Kanagawa
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorders characterized by the selective loss of upper and lower motor neurons. Although the mechanism for the molecular pathogenesis of ALS is still unclear, motor neuron disease researches have supported that oxidative stress is strongly implicated in ALS pathology. We previously identified NDDPX08, which is being used in another neurodegenerative disease, selectively exerts cell protection against oxidative stress-induced cell death. In this study, we evaluated whether NDDPX08 improved neurological progression in ALSSOD1H46R mouse model. ALS mice were received once-daily intraperitoneal administration of NDDPX08 (0, 1 and 10 mg/kg) after showing a sign of the
P2-o04 The investigation into the degradation of the Notch3 protein in the stable cell lines expressing the wild and mutant Notch3 Akiko Hosomi 1 , Toshiki Mizuno 1 , Yoshihisa Watanabe 2 , Masaki Tanaka 2 , Masanori Nakagawa 1 1
Department of Neurology, Kyoto Prefectural University of Medicine, Kyoto Department of Cell Biology Research Institute for Neurological Disease and Geriatrics 2
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) is caused by a point mutation that encodes the extracellular domain of NOTCH3 (ECD-N3). It has not been known how CADASIL mutated N3 lead to the degeneration of vascular smooth muscle cells. Previous reports show that the ECD-N3 mutant protein abnormally accumulated in the arteries from the CADASIL patients. We assume that the proteolysis of ECD-N3 or the N3-processing pathway is impaired by the mutations. To address this hypothesis, we generated stable cell lines expressing the mutant (C185R) or the wild type N3 using the Flp-In T-Rex system. A single copy of these vectors was integrated at the FRT site in Flp-In T-Rex 293 host cell, and the expression of N3 was regulated by the addition of tetracycline. To determine the half-life of each N3 protein, these stable cells were grown in the absence of tetracycline for 0–7days. The half-lives of full length, ECD or intracellular domain N3 proteins were measured by western blotting analysis. There was no difference between the degradation rates of wild–type and C185R-mutant N3. However, immunocytochemical analysis of these stable cells identified that both N3 protein, in part, accumulated intracellularly. We then measured the degradation of N3 protein on cell surface by biotinylation experiment. We will discuss the relationship between CADASIL mutation and N3 protein metabolism. doi:10.1016/j.neures.2010.07.1371
P2-o05 Protective effect of hesperidin and naringin against 3 nitropropionic acid induced Huntingtons like symptoms in rats possible role of nitric oxide Puneet Kumar , Anil Kumar Pharmacology, Panjab University 3-nitropropionic acid (3-NP) is a well known experimental model to study Huntingtons disease (HD) and associated neuropsychiatric problems. Present study has been designed to explore the protective effects of hesperidin, naringin, and their nitric oxide mechanism (if any) against 3-nitropropionic acid induced neurotoxicity in rats. Systemic 3-nitropropionic acid (10 mg/kg) treatment for 14 days in rats significantly induced HD like symptoms in rats as indicated by reduced locomotor activity, body weight, grip strength, oxidative defense and mitochondrial complex enzymes (complex I, II, and IV) activities in striatum. Naringin and hesperidin pretreatment significantly attenuated behavioural alterations, oxidative stress and mitochondrial enzyme complex dysfunction in 3-NP treated group. l-arginine (50 mg/kg) pretreatment with lower dose of hesperidin (50 mg/kg) and naringin (50 mg/kg) significantly attenuated their protective effect respectively. Whereas l-NAME (10 mg/kg), a non selective NOS inhibitor pretreatment with hesperidin (50 mg/kg) and naringin (50 mg/kg) significantly potentiated their protective effect which was significant as compared to their effect per se. Study highlights the therapeutic potential of hesperidin and naringin against Huntingtons like conditions and further indicates that these drugs might act through nitric oxide mechanism. doi:10.1016/j.neures.2010.07.1372
P2-o06 Melanin-concentrating hormone neurons, rather than orexin neurons, are vulnerable to quinolinate cytotoxicity in vivo Kanae Obukuro , Moeko Takigawa, Akinori Hisatsune, Yoichiro Isohama, Hiroshi Katsuki Department of Chemico-Pharmacological Sciences, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto Orexins are neuropeptides produced in the lateral hypothalamus (LH) that play an important role in regulation of sleep–wake cycle. Several studies indicate that selective loss of orexin neurons causes narcolepsy, but the mechanisms of this pathological change remain unclear. A previous study showed that quinolinic acid (QA), acting as an endogenous agonist at N-methyl-d-aspartate (NMDA) receptors, caused selective loss of orexin neurons in rat hypothalamic slice culture, with melanin-concentrating hormone (MCH) neurons preserved. Here we examined QA cytotoxicity in vivo.
e309
QA was injected into LH of adult C57BL/6 mice, and frozen brain sections were obtained three days later for double immunofluorescence staining of orexin and MCH. The number of positive cells of all sections of QA-injected side was compared with control side. Unexpectedly, injection of QA (60 and 120 nmol) caused a selective and dose-dependent decrease of the number of MCH neurons while having no significant effect on the number of orexin neurons. This toxicity was attenuated by 10 nmol MK-801, an NMDA receptor antagonist. Next we examined whether QA cytotoxicity could be affected when GABAergic inhibitory inputs were shut off. Injection of QA (60 nmol) with GABAA receptor antagonists such as bicuculline (1 nmol) and picrotoxin (2 nmol) again caused selective loss of MCH neurons as compared to orexin neurons. A significant decrease in the number of orexin neurons was induced when QA injection was performed in the dark phase of diurnal cycle (14.8% and 21.8% reduction by 60 and 120 nmol QA respectively), but the degree of the decrease was still lower than that in the number of MCH neurons (40.8% and 57.4% reduction by 60 and 120 nmol QA respectively). Finally, selective loss of MCH neurons by injection of QA (60 nmol) was also observed in Wistar rats at 3–4 weeks of age. These results do not support the hypothesis that acute excitotoxicity is responsible for the pathogenesis of narcolepsy. doi:10.1016/j.neures.2010.07.1373
P2-o07 The localization of the C terminal fragment of ␣1A calcium channel influences the CREB target transcription Makoto Takahashi , Kinya Ishikawa, Tarou Ishiguro, Masato Obayashi, Hidehiro Mizuisawa Dept of Neurology and Neurological Science, Tokyo Medical and Dental University, Tokyo Background: Spinocerebellar ataxia type6 (SCA6) is considered one of nine polyglutamine diseases, which is caused by the polyglutamine(polyQ) repeat expansion, and the cause of SCA6 is the polyQ repeat expansion of the Carboxyl(C) terminal of ␣1A voltage-gated calcium channel(Cav2.1), but the basic mechanism for neurodegeneration has been unknown. The C terminal fragment (CTF) of Cav2.1,which may be cleaved from the full length Cav2.1, basically locates in the cytoplasm, but recent reports found the possibility that CTF is tend to locate in the nucleus, when the polyQ length of CTF are expanded. Object: To examine whether the location of CTF of Cav2.1 influences the transcription machinery and pathomechanism of SCA6. Method: At first, we made the various polyQ length of CTF vectors, with nuclear export signal (NES) or nuclear localization signal (NLS). With transient expression cell models, we checked the influence of CTF for CREB target transcripition with the use of CRE-luciferase assay and GAL4-CREB-luciferase assay. Result: When the CTF of Cav2.1 locates in the nucleus, CREB target transcription are suppressed and when they locate in the cytoplasm, CREB target transcription are increased. As the polyQ length expands, the transcription activity is gradually increased, but when the polyQ length is too long, the transcription is suppressed. Conclusion: The localization and polyQ length of CTF of Cav2.1 may influence the CREB target transcription. doi:10.1016/j.neures.2010.07.1374
P2-o08 The therapeutic agent NDDPX08 promotes sustainable motor performance and extends a post-onset survival interval in an ALS mouse model Kazunori Tanaka 1 , Takuya Kanno 2 , Yoshiko Yanagisawa 2 , Masashi Aoki 3 , Shinji Hadano 2 , Yasuto Itoyama 3 , Mieko Ogino 4 , Yasuo Iwasaki 5 , Fumihito Yoshii 6 , Joh-E. Ikeda 1,2 1
Neugen Pharma Inc. Kanagawa 2 Dept of Mol Life Sci, Tokai Univ Scl of Med, Kanagawa 3 Dept of Neurol, Tohoku Univ Grad Sch of Med, Miyagi 4 Dept of Neurol, Kitasato Univ East Hospital, Kanagawa 5 Dept of Neurol, Toho Univ Omori Hospital, Tokyo 6 Dept of Neurol, Tokai Univ Sch of Med, Kanagawa
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorders characterized by the selective loss of upper and lower motor neurons. Although the mechanism for the molecular pathogenesis of ALS is still unclear, motor neuron disease researches have supported that oxidative stress is strongly implicated in ALS pathology. We previously identified NDDPX08, which is being used in another neurodegenerative disease, selectively exerts cell protection against oxidative stress-induced cell death. In this study, we evaluated whether NDDPX08 improved neurological progression in ALSSOD1H46R mouse model. ALS mice were received once-daily intraperitoneal administration of NDDPX08 (0, 1 and 10 mg/kg) after showing a sign of the