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Impact of proteasomal inhibition and formation of ubiquitinated inclusions on cultured neurons from SAMP10 mice
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Abstracts / Neuroscience Research 58S (2007) S1–S244
P1-h39 Impact of proteasomal inhibition and formation of ubiquitinated inclusions on cultured neurons from SAMP10 mice Yoichi Chiba, Atsuyoshi Shimada, Naoko Kumagai, Noriko Kawamura, Keisuke Yoshikawa, Sanae Ishii, Ayako Furukawa, Masanori Hosokawa Department of Pathology, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Japan SAMP10 mouse (P10) is a model for age-associated neurodegeneration. We have reported an age-related increase in neuronal cytoplasmic ubiquitin-positive inclusions (UPIs) and concomitant decrease in proteasomal activity in the limbic-related structures of P10 brain. To study the effect of decreased proteasomal activity and formation of UPIs on neuronal survival and morphology, we performed in vitro proteasome inhibition experiment using cultured cortical neurons from P10 and normal aging control SAMR1 mice (R1). Significant cell death occured 24 h after the addition of 1–10 M MG115, a potent proteasome inhibitor, with no strain difference. UPIs, formed by exposing cells to 3 M MG115 for 24 h, were more frequently observed in P10 neurons than in R1 neurons. Morphometric analysis revealed that UPI-bearing neurons had fewer long (>50 m) neurites when compared to those without UPIs. UPIs may exert adverse effects on the maintenance of dendritic arbors.
P1-h44 A drosophila PQBP-1 mutant is a novel model for mental retardation Takuya Tamura, Daisuke Horiuchi, Hitoshi Okazawa Department of Neuropathology, MRI, Tokyo Medical and Dental University, Japan Recently, it has been reported that mutations of polyglutamine tract binding protein-1 (PQBP-1) are responsible for mental retardation. We found a piggyBack inserted fly is hypomorph mutant of PQBP-1. We examined whether the PQBP-1 mutant shows a defect in learning and memory and other phenotypes. Memory acquisition but not maintenance was disrupted during a classical olfactory conditioning in a PQBP-1 mutant fly. We observed loss of projection neurons which are related to early phase memory. Life-span of the mutant was shorter than wild type. The mutant fly showed enhanced courtship behavior, and a little disorder of circadian rhythms. Moreover, expression of exogenous PQBP-1 rescued the learning defect. Then, we conclude that the mutant is a novel mental retardation model. Next, we screened chemicals to recover the phenotypes of the mutant. We report results of the chemical test.
P1-h41
Vulnerability to excitotoxic insults and defective microglial response in SAMP10 mouse
P1-h45 Effects of mutant ␥PKC found in SCA14 on the nature
Atsuyoshi Shimada, Sanae Ishii, Keisuke Yoshikawa, Yoichi Chiba, Noriko Kawamura, Naoko Kumagai, Ayako Furukawa, Masanori Hosokawa Department of Pathology, Institute for Developmental Research, Aichi Human Service Center, Kaugai, Japan
Takahiro Seki 1 , Takayuki Shimahara 1 , Nana Abe 1 , Kazuhiro Yamamoto 1 , Naoko Adachi 2 , Kaori Kashiwagi 2 , Naoaki Saito 2 , Norio Sakai 1 1 Department of Molecular Pharmacology and Neuroscience, Graduate School of Biomedical Sciences, Hiroshima University, Japan; 2 Laboratory of Molecular Pharmacology, Biosignal Research Centre, Kobe University, Japan
Microglia may play a pivotal role for neuroprotection. We studied glial responses to excitotoxic insults in SAMP10 mice (P10), a model of agerelated cerebral degeneration and normal control SAMR1 mice (R1). Three-month-old P10 and R1 received kainate (KA) intraperitoneally. Three days later, brains were stained with cresyl violet and immunohistochemically (Iba1). Hippocampal damages were graded as Grade 3 (widespread neuronal death), 2 (focal neuronal death) and 1 (no cell death but only glial response). Of 7 KA-treated P10, one, five and one had Grade 1, 2 and 3 lesions, respectively. All 10 KA-treated R1 had Grade 1. Many microglia had hypertrophic somata and bushy processes in Grade 1 lesions of R1. The cell-to-cell contact with neurons was enhanced in these hypertrophic microglia. Microglia in Grade 1 and 2 lesions of P10 lacked hypertrophy or bushy processes. The defective response of microglia may be causally related to neuronal vulnerability in P10. Research fund: KAKENHI 18590396
of primary cultured Purkinje cells
Recently, missense mutations have been identified in protein kinase C␥ (␥PKC) gene in several families of spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disorder. We have previously demonstrated that these mutant ␥PKC are susceptible to cytoplasmic aggregation and causes cell death in CHO cells, indicating that this property is involved in the etiology of SCA14. In the present study, we examined how mutant ␥PKC affect the nature of Purkinje cells (PCs), which are degenerated in SCA14 patients, in mouse cerebellar primary culture. Mutant ␥PKC-GFP frequently formed aggregates in PCs. These aggregates were ubiquitin-positive and colocalized with proteasome. Moreover, mutant ␥PKC-GFPs significantly reduced the dendritic areas and the density of dendritic spines of PCs. These results indicate that mutant ␥PKC not only forms aggregates but also affects the dendritic morphology and the synaptic formation in PCs. Research fund: KAKENHI 17700351
P1-h42 Effect of semi-fluid diet breeding on the hippocampal dopamine release Sachio Kushida 1 , Norio Hori 1 , Katsuhiko Kimoto 1,3 , Minoru Toyoda 1 , Atsumi Nitta 4 , Toshitaka Nabeshima 4 , Minoru Onozuka 2,3 1 Department of Oral & Maxillofacial Rehabilitation, Kanagawa dental college, Kanagawa, Japan; 2 Department of Functional Biology, Physiology & Neuroscience, Kanagawa Dental College. Kanagawa, Japan; 3 Research Center of Brain and Oral Science, Kanagawa Dental College, Kanagawa, Japan; 4 Department of Neuropsychopharmacology and Hospital Pharmacy, Nagoya University Graduate School of Medicine, Aichi, Japan Using the intracerebral microdialysis technique, we examined whether the semi-fluid diet breeding is involved in the dopaminergic neuronal systems in the rat hippocampus. The hippocampal dopamine release under the high K+ condition was analyzed in four groups: (1) Normal group fed with a hard food, (2) Normal group fed with soft food, (3) AD group (infusing the Beta-amyloid protein) fed with hard food, (4) AD group fed with soft food. In all groups, increases in the extracellular levels of dopamine (DA) were seen; however, the DA levels of the normal and AD groups with soft food were significantly lower than those of hard food groups. The data suggest the link between hardness of food and hippocampal DA processes.
P1-h46 Possible roles of LC3, ubiquitin, and p62 in the accumulation of lysosomes in neurons deficient in cathepsin D Masato Koike 1 , Masahiro Shibata 1 , Tetsuro Ishii 2 , Yasuo Uchiyama 1 1 Department of Cell Biology and Neurosciences, Osaka University, Suita, Osaka, Japan; 2 Biomolecular and Integrated Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Ibaraki, Japan We have shown that autophagy contributes to the accumulation of lysosomal structures such as granular osmiophilic deposits (GRODs) in CNS tissue of cathepsin cathepsin D deficient (CD−/−) mice, a mouse model of neuronal ceroid lipofuscinosis (Koike et al., 2000, 2005). Using immunoelectron microscopy we found that ubiquitin, together with p62/A170/SQSTM1, a polyubiquitin binding protein, was localized on the surface membrane of GRODs in CD−/− neurons. Since P62 has been shown to interact also with LC3, a marker protein of autophagosome (Bjorkoy et al., 2005), we confirmed the co-existence of these three molecules. Based upon these findings we propose a model whereby p62 interacts with ubiquitin, as aggregates in the cytosol, on the membrane of abnormal organelles such as GRODs, and further interact with LC3 to degrade them by forming autophagosomes. Research fund: KAKENHI (17790141, 18021019)
Abstracts / Neuroscience Research 58S (2007) S1–S244
P1-h39 Impact of proteasomal inhibition and formation of ubiquitinated inclusions on cultured neurons from SAMP10 mice Yoichi Chiba, Atsuyoshi Shimada, Naoko Kumagai, Noriko Kawamura, Keisuke Yoshikawa, Sanae Ishii, Ayako Furukawa, Masanori Hosokawa Department of Pathology, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Japan SAMP10 mouse (P10) is a model for age-associated neurodegeneration. We have reported an age-related increase in neuronal cytoplasmic ubiquitin-positive inclusions (UPIs) and concomitant decrease in proteasomal activity in the limbic-related structures of P10 brain. To study the effect of decreased proteasomal activity and formation of UPIs on neuronal survival and morphology, we performed in vitro proteasome inhibition experiment using cultured cortical neurons from P10 and normal aging control SAMR1 mice (R1). Significant cell death occured 24 h after the addition of 1–10 M MG115, a potent proteasome inhibitor, with no strain difference. UPIs, formed by exposing cells to 3 M MG115 for 24 h, were more frequently observed in P10 neurons than in R1 neurons. Morphometric analysis revealed that UPI-bearing neurons had fewer long (>50 m) neurites when compared to those without UPIs. UPIs may exert adverse effects on the maintenance of dendritic arbors.
P1-h44 A drosophila PQBP-1 mutant is a novel model for mental retardation Takuya Tamura, Daisuke Horiuchi, Hitoshi Okazawa Department of Neuropathology, MRI, Tokyo Medical and Dental University, Japan Recently, it has been reported that mutations of polyglutamine tract binding protein-1 (PQBP-1) are responsible for mental retardation. We found a piggyBack inserted fly is hypomorph mutant of PQBP-1. We examined whether the PQBP-1 mutant shows a defect in learning and memory and other phenotypes. Memory acquisition but not maintenance was disrupted during a classical olfactory conditioning in a PQBP-1 mutant fly. We observed loss of projection neurons which are related to early phase memory. Life-span of the mutant was shorter than wild type. The mutant fly showed enhanced courtship behavior, and a little disorder of circadian rhythms. Moreover, expression of exogenous PQBP-1 rescued the learning defect. Then, we conclude that the mutant is a novel mental retardation model. Next, we screened chemicals to recover the phenotypes of the mutant. We report results of the chemical test.
P1-h41
Vulnerability to excitotoxic insults and defective microglial response in SAMP10 mouse
P1-h45 Effects of mutant ␥PKC found in SCA14 on the nature
Atsuyoshi Shimada, Sanae Ishii, Keisuke Yoshikawa, Yoichi Chiba, Noriko Kawamura, Naoko Kumagai, Ayako Furukawa, Masanori Hosokawa Department of Pathology, Institute for Developmental Research, Aichi Human Service Center, Kaugai, Japan
Takahiro Seki 1 , Takayuki Shimahara 1 , Nana Abe 1 , Kazuhiro Yamamoto 1 , Naoko Adachi 2 , Kaori Kashiwagi 2 , Naoaki Saito 2 , Norio Sakai 1 1 Department of Molecular Pharmacology and Neuroscience, Graduate School of Biomedical Sciences, Hiroshima University, Japan; 2 Laboratory of Molecular Pharmacology, Biosignal Research Centre, Kobe University, Japan
Microglia may play a pivotal role for neuroprotection. We studied glial responses to excitotoxic insults in SAMP10 mice (P10), a model of agerelated cerebral degeneration and normal control SAMR1 mice (R1). Three-month-old P10 and R1 received kainate (KA) intraperitoneally. Three days later, brains were stained with cresyl violet and immunohistochemically (Iba1). Hippocampal damages were graded as Grade 3 (widespread neuronal death), 2 (focal neuronal death) and 1 (no cell death but only glial response). Of 7 KA-treated P10, one, five and one had Grade 1, 2 and 3 lesions, respectively. All 10 KA-treated R1 had Grade 1. Many microglia had hypertrophic somata and bushy processes in Grade 1 lesions of R1. The cell-to-cell contact with neurons was enhanced in these hypertrophic microglia. Microglia in Grade 1 and 2 lesions of P10 lacked hypertrophy or bushy processes. The defective response of microglia may be causally related to neuronal vulnerability in P10. Research fund: KAKENHI 18590396
of primary cultured Purkinje cells
Recently, missense mutations have been identified in protein kinase C␥ (␥PKC) gene in several families of spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disorder. We have previously demonstrated that these mutant ␥PKC are susceptible to cytoplasmic aggregation and causes cell death in CHO cells, indicating that this property is involved in the etiology of SCA14. In the present study, we examined how mutant ␥PKC affect the nature of Purkinje cells (PCs), which are degenerated in SCA14 patients, in mouse cerebellar primary culture. Mutant ␥PKC-GFP frequently formed aggregates in PCs. These aggregates were ubiquitin-positive and colocalized with proteasome. Moreover, mutant ␥PKC-GFPs significantly reduced the dendritic areas and the density of dendritic spines of PCs. These results indicate that mutant ␥PKC not only forms aggregates but also affects the dendritic morphology and the synaptic formation in PCs. Research fund: KAKENHI 17700351
P1-h42 Effect of semi-fluid diet breeding on the hippocampal dopamine release Sachio Kushida 1 , Norio Hori 1 , Katsuhiko Kimoto 1,3 , Minoru Toyoda 1 , Atsumi Nitta 4 , Toshitaka Nabeshima 4 , Minoru Onozuka 2,3 1 Department of Oral & Maxillofacial Rehabilitation, Kanagawa dental college, Kanagawa, Japan; 2 Department of Functional Biology, Physiology & Neuroscience, Kanagawa Dental College. Kanagawa, Japan; 3 Research Center of Brain and Oral Science, Kanagawa Dental College, Kanagawa, Japan; 4 Department of Neuropsychopharmacology and Hospital Pharmacy, Nagoya University Graduate School of Medicine, Aichi, Japan Using the intracerebral microdialysis technique, we examined whether the semi-fluid diet breeding is involved in the dopaminergic neuronal systems in the rat hippocampus. The hippocampal dopamine release under the high K+ condition was analyzed in four groups: (1) Normal group fed with a hard food, (2) Normal group fed with soft food, (3) AD group (infusing the Beta-amyloid protein) fed with hard food, (4) AD group fed with soft food. In all groups, increases in the extracellular levels of dopamine (DA) were seen; however, the DA levels of the normal and AD groups with soft food were significantly lower than those of hard food groups. The data suggest the link between hardness of food and hippocampal DA processes.
P1-h46 Possible roles of LC3, ubiquitin, and p62 in the accumulation of lysosomes in neurons deficient in cathepsin D Masato Koike 1 , Masahiro Shibata 1 , Tetsuro Ishii 2 , Yasuo Uchiyama 1 1 Department of Cell Biology and Neurosciences, Osaka University, Suita, Osaka, Japan; 2 Biomolecular and Integrated Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Ibaraki, Japan We have shown that autophagy contributes to the accumulation of lysosomal structures such as granular osmiophilic deposits (GRODs) in CNS tissue of cathepsin cathepsin D deficient (CD−/−) mice, a mouse model of neuronal ceroid lipofuscinosis (Koike et al., 2000, 2005). Using immunoelectron microscopy we found that ubiquitin, together with p62/A170/SQSTM1, a polyubiquitin binding protein, was localized on the surface membrane of GRODs in CD−/− neurons. Since P62 has been shown to interact also with LC3, a marker protein of autophagosome (Bjorkoy et al., 2005), we confirmed the co-existence of these three molecules. Based upon these findings we propose a model whereby p62 interacts with ubiquitin, as aggregates in the cytosol, on the membrane of abnormal organelles such as GRODs, and further interact with LC3 to degrade them by forming autophagosomes. Research fund: KAKENHI (17790141, 18021019)