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p-Nitrotoluene models of rat Parkinsonism
Abstracts ceptible to various toxins and this indicated this region was an equivalent of human substantia nigra. Conclusion Medaka is an attractive model animal for PD research. doi:10.1016/j.neures.2009.09.557
Dept. Neurol., Univ. of Nagoya, Nagoya, Japan; 2 Ins. Adv. Res., Univ. of Nagoya, Nagoya, Japan; 3 Dept. Cell. Pharmacol., Univ. of Nagoya, Nagoya, Japan; 4 Stroke Center, Aichi Med. Univ., Japan
National Institute for Environmental Studies, Tsukuba, Japan Sporadic neurodegenerative diseases have documented environmental causes. An animal model of Parkinson’s disease has been developed by selective dopaminergic degeneration with a pesticide, rotenone in adult animals. Since the hypothesis of DOHaD (Developmental origins of health and disease) has been proposed, we examined the behavioral effects of p-nitrotoluene, which caused developmental deficit of dopaminergic tone. p-Nitrotoluene (3 mg/kg/day) was subcutaneously infused to 7-week-old male Wistar rats, using Alzet minipumps. The spontaneous motor activity was 12% less than that of control rat, suggesting that the chemical caused hypoactivity, as seen in the rotenone model of Parkinson’s disease. Thus, the data suggest that a single environmental chemical could develop different disorders in rats, dependently of exposure periods. doi:10.1016/j.neures.2009.09.558
2
Clin.
We examined the involvement of endogenous dopamine and proteasome activity in paraquat toxicity. Depletion of endogenous dopamine by ␣-methyl-p-tyrosine (␣-MT) protected PC12 cells from paraquat toxicity. The effects of paraquat on proteasome activity were examined in PC12 cells overexpressing a proteasome sensitive green fluorescent protein. Paraquat increased the number of propidium iodide (PI)negative cells with low proteasome activity and that of PI(+) cells. Pre-treatment with ␣-MT did not alter the number of PI(−) cells with low proteasome activity, but reduce that of PI(+) cells. Simultaneous treatment with MG-132, a proteasome inhibitor, accelerated an increase in the number of PI(−) cells with low proteasome activity and reduced that of PI(+) cells. These results suggest that reduction of proteasome activity may be involved in cellular defense mechanisms against dopamine-mediated paraquat toxicity. doi:10.1016/j.neures.2009.09.559
P1-k18 Immunoisolation of striatal dopaminergic nerve terminals defines susceptibility to MPTP toxicity in C57BL/6 mice Shota Kobayashi, Akiko Muroyama, Hiroaki Matsushima, Yasuhide Mitsumoto Dept. of Alternative Med. and Exp. Therapeutics, Faculty of Pharmaceutical Sciences, Hokuriku University, Japan 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is well known as a Parkinson’s disease-inducing toxin. But the precise mechanism underlying MPTP neurotoxicity is not fully understood. In the present study, we tried to isolate striatal dopaminergic nerve terminals using a immuno-magnetic protocol with a polyclonal antibody against the second extracellular loop of dopamine transporter (DAT) and investigated MPTP-induced degeneration of striatal dopaminergic neuron in C57BL/6 mice. Striatal synaptosomes were prepared from vehicle- and MPTP-treated mice. The fraction isolated from the synaptosomes by immunoisolation technique subjected to western analysis to assess relative dopaminergic versus non-dopaminergic synaptosomal purification using antibodies against-DAT, glutamic acid decarboxylase or synaptophysin as a housekeeping protein. We show that this technique enables to isolate the dopaminergic synaptosomes from the striatum, and the specificity of MPTP toxicity can be assessed precisely. doi:10.1016/j.neures.2009.09.560
TDP-43 is known to be a major component of the ubiquitinated inclusions characteristic of ALS and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Although TDP-43 is a nuclear protein, it disappears from the nucleus of affected neurons and glial cells, implicating TDP-43 loss-of-function in the pathogenesis of neurodegeneration. Here we show that the knock-down of TDP-43 in differentiated Neuro-2a cells inhibited neurite outgrowth and induced cell death. In knocked-down cells, the Rho family members RhoA, Rac1, and Cdc42 GTPases were inactivated. In addition, TDP-43 depletion significantly suppressed protein geranylgeranylation. These data suggest that TDP-43 plays a key role in the regulation of Rho family GTPases through protein geranylgeranylation. We conclude that the lossof-function of TDP-43 appears to underlie the pathogenesis of neurodegeneration in TDP-43 proteinopathies. doi:10.1016/j.neures.2009.09.561
P1-k17 Involvement of endogenous dopamine in paraquat-induced cytotoxicity: Cell fate decisions by proteasome activity Yasuhiko Izumi 1 , Noriyuki Yamamoto 1 , Sayaka Matsushima 1 , Takaaki Matsuo 1 , Seiko Wakita 1 , Toshiaki Kume 1 , Hideyuki Sawada 2 , Akinori Akaike 1 Dept. Pharmacol., Grad. Sch. Pharm. Sci., Kyoto Univ, Kyoto, Japan; Res. Cent., Utano Nat. Hosp., Kyoto, Japan
P1-k19 TDP-43 depletion induces neuronal cell damage through dysregulation of Rho family GTPases Yohei Iguchi 1 , Masahisa Katsuno 2 , Jun-Ichi Niwa 4 , Jun Sone 1 , Adachi 1 , Fumiaki Tanaka 1 , Kozo Masahiro Waza 1 , Hiroaki Kaibuchi 3 , Gen Sobue 1 1
P1-k16 p-Nitrotoluene models of rat Parkinsonism Junko Suzuki, Masami Ishido
1
S119
P1-k20 Constitutive dimer formation of TDP-43 in human cell lines Jun-ichi Satoh, Yuki Shiina, Hiroko Tabunoki Dept. Bioinformatics, Meiji Pharm. Univ., Tokyo, Japan Ubiquitinated and abnormally phosphorylated TDP-43 is identified as a principal component of neuronal and glial inclusions in FTLD and ALS. In TDP-43 proteinopathy, it forms cytoplasmic aggregates in contrast to the normal nuclear localization, although the mechanism for the nuclear-cytoplasmic shuttling remains unknown. To identify TDP-43-binding proteins involved in its intracellular transport, Flag-tagged TDP-43 was expressed in HEK293, HeLa, and SK-N-SH cells. They were processed for immunoprecipitation with anti-Flag antibody agarose. In all cell lines, we identified endogenous TDP-43 as one of the proteins associated with Flag-tagged TDP-43 but not with Flag-tagged GFP. We identified the C-terminal segment spanning aa 261414 as a domain essential for dimerizaion of TDP-43. Furthermore, we detected a TDP-43 dimer in homogenate of human brain tissues. The homodimer was recovered chiefly form the cytosol fraction. The levels of constitutive TDP-43 dimer formation were unaffected by treatment with MG-132. TDP-43 dimer might serve as a seed that promotes its aggregation in FTLD and ALS. doi:10.1016/j.neures.2009.09.562
P1-l01 The spinal anterior horn has the capacity to self-regenerate in ALS model mice Kazunori Miyazaki, Makiko Nagai, Nobutoshi Morimoto, Tomoko Kurata, Yasushi Takehisa, Yoshio Ikeda, Koji Abe Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Science, Japan The exact host environment necessary for neural regeneration in amyotrophic lateral sclerosis (ALS) has not yet been fully elucidated. We first focused on the extracellular matrix proteins in ALS model mice during the development of the disease and then attempted to examine whether regeneration occurs in the ALS spinal cord. A progressive increase in ␥1laminin (a promoter of regeneration) and a progressive decrease in semaphorin3A (Sema3A) (an inhibitor of regeneration) were observed, mainly in the neuropil of the spinal anterior horn, when astrocytes began to express both ␥1 laminin and Sema3A. On the other hand, a progressive increase in growth associated protein 43 (GAP43) (synaptic regeneration site) and a progressive decrease in synaptotagmin1 (actual synaptic bouton) were observed in the same areas of the spinal anterior horn. Thus, the present data suggest that although the spinal anterior horn in ALS models loses motor neurons, it initially possesses the capacity to self-regenerate. doi:10.1016/j.neures.2009.09.563
Dept. Neurol., Univ. of Nagoya, Nagoya, Japan; 2 Ins. Adv. Res., Univ. of Nagoya, Nagoya, Japan; 3 Dept. Cell. Pharmacol., Univ. of Nagoya, Nagoya, Japan; 4 Stroke Center, Aichi Med. Univ., Japan
National Institute for Environmental Studies, Tsukuba, Japan Sporadic neurodegenerative diseases have documented environmental causes. An animal model of Parkinson’s disease has been developed by selective dopaminergic degeneration with a pesticide, rotenone in adult animals. Since the hypothesis of DOHaD (Developmental origins of health and disease) has been proposed, we examined the behavioral effects of p-nitrotoluene, which caused developmental deficit of dopaminergic tone. p-Nitrotoluene (3 mg/kg/day) was subcutaneously infused to 7-week-old male Wistar rats, using Alzet minipumps. The spontaneous motor activity was 12% less than that of control rat, suggesting that the chemical caused hypoactivity, as seen in the rotenone model of Parkinson’s disease. Thus, the data suggest that a single environmental chemical could develop different disorders in rats, dependently of exposure periods. doi:10.1016/j.neures.2009.09.558
2
Clin.
We examined the involvement of endogenous dopamine and proteasome activity in paraquat toxicity. Depletion of endogenous dopamine by ␣-methyl-p-tyrosine (␣-MT) protected PC12 cells from paraquat toxicity. The effects of paraquat on proteasome activity were examined in PC12 cells overexpressing a proteasome sensitive green fluorescent protein. Paraquat increased the number of propidium iodide (PI)negative cells with low proteasome activity and that of PI(+) cells. Pre-treatment with ␣-MT did not alter the number of PI(−) cells with low proteasome activity, but reduce that of PI(+) cells. Simultaneous treatment with MG-132, a proteasome inhibitor, accelerated an increase in the number of PI(−) cells with low proteasome activity and reduced that of PI(+) cells. These results suggest that reduction of proteasome activity may be involved in cellular defense mechanisms against dopamine-mediated paraquat toxicity. doi:10.1016/j.neures.2009.09.559
P1-k18 Immunoisolation of striatal dopaminergic nerve terminals defines susceptibility to MPTP toxicity in C57BL/6 mice Shota Kobayashi, Akiko Muroyama, Hiroaki Matsushima, Yasuhide Mitsumoto Dept. of Alternative Med. and Exp. Therapeutics, Faculty of Pharmaceutical Sciences, Hokuriku University, Japan 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is well known as a Parkinson’s disease-inducing toxin. But the precise mechanism underlying MPTP neurotoxicity is not fully understood. In the present study, we tried to isolate striatal dopaminergic nerve terminals using a immuno-magnetic protocol with a polyclonal antibody against the second extracellular loop of dopamine transporter (DAT) and investigated MPTP-induced degeneration of striatal dopaminergic neuron in C57BL/6 mice. Striatal synaptosomes were prepared from vehicle- and MPTP-treated mice. The fraction isolated from the synaptosomes by immunoisolation technique subjected to western analysis to assess relative dopaminergic versus non-dopaminergic synaptosomal purification using antibodies against-DAT, glutamic acid decarboxylase or synaptophysin as a housekeeping protein. We show that this technique enables to isolate the dopaminergic synaptosomes from the striatum, and the specificity of MPTP toxicity can be assessed precisely. doi:10.1016/j.neures.2009.09.560
TDP-43 is known to be a major component of the ubiquitinated inclusions characteristic of ALS and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Although TDP-43 is a nuclear protein, it disappears from the nucleus of affected neurons and glial cells, implicating TDP-43 loss-of-function in the pathogenesis of neurodegeneration. Here we show that the knock-down of TDP-43 in differentiated Neuro-2a cells inhibited neurite outgrowth and induced cell death. In knocked-down cells, the Rho family members RhoA, Rac1, and Cdc42 GTPases were inactivated. In addition, TDP-43 depletion significantly suppressed protein geranylgeranylation. These data suggest that TDP-43 plays a key role in the regulation of Rho family GTPases through protein geranylgeranylation. We conclude that the lossof-function of TDP-43 appears to underlie the pathogenesis of neurodegeneration in TDP-43 proteinopathies. doi:10.1016/j.neures.2009.09.561
P1-k17 Involvement of endogenous dopamine in paraquat-induced cytotoxicity: Cell fate decisions by proteasome activity Yasuhiko Izumi 1 , Noriyuki Yamamoto 1 , Sayaka Matsushima 1 , Takaaki Matsuo 1 , Seiko Wakita 1 , Toshiaki Kume 1 , Hideyuki Sawada 2 , Akinori Akaike 1 Dept. Pharmacol., Grad. Sch. Pharm. Sci., Kyoto Univ, Kyoto, Japan; Res. Cent., Utano Nat. Hosp., Kyoto, Japan
P1-k19 TDP-43 depletion induces neuronal cell damage through dysregulation of Rho family GTPases Yohei Iguchi 1 , Masahisa Katsuno 2 , Jun-Ichi Niwa 4 , Jun Sone 1 , Adachi 1 , Fumiaki Tanaka 1 , Kozo Masahiro Waza 1 , Hiroaki Kaibuchi 3 , Gen Sobue 1 1
P1-k16 p-Nitrotoluene models of rat Parkinsonism Junko Suzuki, Masami Ishido
1
S119
P1-k20 Constitutive dimer formation of TDP-43 in human cell lines Jun-ichi Satoh, Yuki Shiina, Hiroko Tabunoki Dept. Bioinformatics, Meiji Pharm. Univ., Tokyo, Japan Ubiquitinated and abnormally phosphorylated TDP-43 is identified as a principal component of neuronal and glial inclusions in FTLD and ALS. In TDP-43 proteinopathy, it forms cytoplasmic aggregates in contrast to the normal nuclear localization, although the mechanism for the nuclear-cytoplasmic shuttling remains unknown. To identify TDP-43-binding proteins involved in its intracellular transport, Flag-tagged TDP-43 was expressed in HEK293, HeLa, and SK-N-SH cells. They were processed for immunoprecipitation with anti-Flag antibody agarose. In all cell lines, we identified endogenous TDP-43 as one of the proteins associated with Flag-tagged TDP-43 but not with Flag-tagged GFP. We identified the C-terminal segment spanning aa 261414 as a domain essential for dimerizaion of TDP-43. Furthermore, we detected a TDP-43 dimer in homogenate of human brain tissues. The homodimer was recovered chiefly form the cytosol fraction. The levels of constitutive TDP-43 dimer formation were unaffected by treatment with MG-132. TDP-43 dimer might serve as a seed that promotes its aggregation in FTLD and ALS. doi:10.1016/j.neures.2009.09.562
P1-l01 The spinal anterior horn has the capacity to self-regenerate in ALS model mice Kazunori Miyazaki, Makiko Nagai, Nobutoshi Morimoto, Tomoko Kurata, Yasushi Takehisa, Yoshio Ikeda, Koji Abe Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Science, Japan The exact host environment necessary for neural regeneration in amyotrophic lateral sclerosis (ALS) has not yet been fully elucidated. We first focused on the extracellular matrix proteins in ALS model mice during the development of the disease and then attempted to examine whether regeneration occurs in the ALS spinal cord. A progressive increase in ␥1laminin (a promoter of regeneration) and a progressive decrease in semaphorin3A (Sema3A) (an inhibitor of regeneration) were observed, mainly in the neuropil of the spinal anterior horn, when astrocytes began to express both ␥1 laminin and Sema3A. On the other hand, a progressive increase in growth associated protein 43 (GAP43) (synaptic regeneration site) and a progressive decrease in synaptotagmin1 (actual synaptic bouton) were observed in the same areas of the spinal anterior horn. Thus, the present data suggest that although the spinal anterior horn in ALS models loses motor neurons, it initially possesses the capacity to self-regenerate. doi:10.1016/j.neures.2009.09.563