- Email: [email protected]
Increased vulnerability to rotenone-induced neurotoxicity in ceruloplasmin-deficient mice
Neuroscience Letters 446 (2008) 56–58
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Increased vulnerability to rotenone-induced neurotoxicity in ceruloplasmin-deficient mice Kazuma Kaneko ∗ , Akiyo Hineno, Kunihiro Yoshida, Shu-ichi Ikeda Department of Neurology and Rheumatology, Shinshu University School of Medicine, 3-1-1 Asahi, 390-8621 Matsumoto, Japan
a r t i c l e
i n f o
Article history: Received 14 July 2008 Received in revised form 15 August 2008 Accepted 30 August 2008 Keywords: Ceruloplasmin Aceruloplasminemia Rotenone Ubiquitin Acrolein Oxidative stress
a b s t r a c t Ceruloplasmin (Cp) is the strongest ferroxidase in human plasma. Hereditary deficiency of this protein, named aceruloplasminemia, is an interesting model to elucidate the pathogenesis and pathophysiology of neurodegeneration induced by oxidative stress. Enhanced oxidative stress due to excessive iron accumulation is observed in the brains of aceruloplasminemia patients. Rotenone, a selective mitochondrial complex I inhibitor, induces neurodegeneration mimicking Parkinson’s disease. We investigated the influence of Cp deficiency upon neurodegeneration using rotenone-treated, Cp-deficient mouse brains. Immunohistochemical examination showed that acrolein, one of the products of lipid peroxides, and ubiquitin were more markedly immunoreacted in the brains of rotenone-treated, Cp-deficient mice than in rotenone-untreated, Cp-deficient or rotenone-treated, wild-type mice. These molecules were localized in neuronal cells. These results suggested that rotenone-induced lipid peroxidation and accumulation of ubiquitin immunoreactivity were enhanced in the absence of Cp. Therefore, Cp may protect neuronal cells from oxidative stress-induced neurodegeneration. © 2008 Elsevier Ireland Ltd. All rights reserved.
Ceruloplasmin (Cp) is a copper-binding ferroxidase that is mainly produced by hepatocytes and secreted into plasma. Cp oxidizes ferrous iron (Fe2+ ) to ferric iron (Fe3+ ) and can facilitate iron loading onto transferrin [3,13]. In the brain, the majority of Cp is derived from astrocytes and is located at the surface of astrocytes as a glycosylphosphatidylinositol (GPI)-anchored form [14]. In the state of Cp deficiency, it is considered that Fe2+ is increased, resulting in an acceleration of hydroxyl radical (HO• ) production in the presence of hydrogen peroxide (H2 O2 ), named the Fenton reaction [3]. Thus, it is likely that oxidative stress is enhanced in Cp-deficient conditions. Aceruloplasminemia, caused by mutations in the Cp gene [2], provides a good model to evaluate the influence of iron-induced oxidative stress upon neurodegeneration. Aceruloplasminemia is characterized clinically by adult-onset diabetes mellitus, retinal degeneration and various neurological symptoms such as involuntary movements, cerebellar ataxia, parkinsonism and dementia [11,18]. The neuropathological findings are summarized as intracytoplasmic iron deposition, and neuronal cell loss [12,8]. Indeed, Jeong and David reported that cerebellar cell loss and iron deposition were increased in aged Cp-deficient mice [5]. Further, we previously reported that the enlarged deformed astrocytes and ubiquitin-positive spheroidal structures are pathological character-
∗ Corresponding author. Tel.: +81 263 37 2673; fax: +81 263 37 3427. E-mail address: [email protected] (K. Kaneko). 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.08.089
istics of the brains of aceruloplasminemia patients [7]. In addition, it has been reported that several markers of oxidative stress, such as malondialdehyde (MDA), 4-hydroxy-2-nonenal (HNE) and carbonylated protein, are significantly elevated in the brains of patients with this disorder [19,6]. These data strongly support the hypothesis that enhanced oxidative stress causes neuronal cell death in aceruloplasminemia. There is evidence that oxidative stress contributes to neuronal dysfunction and death in several neurodegenerative diseases, including Parkinson’s disease [4]. In Parkinson’s disease, reduced activity of the mitochondrial respiratory chain complex I in dopaminergic neurons is one of the factors enhancing oxidative stress [15]. Rotenone, a neurotoxic pesticide, is a selective inhibitor of the mitochondrial respiratory chain complex I, as does 1methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (MPTP). The chronic infusion of rotenone has been shown to induce neurodegeneration or motor dysfunction mimicking parkinsonian features in the rat or mouse [1,16]. Ubiquitin, which has a central role in protein degradation [9], is overexpressed in the brains of these animals, suggesting the accumulation of damaged proteins targeted for proteasomal degradation. The presence of ubiquitin-positive structures in the brains of aceruloplasminemia patients indicates that the ubiquitin–proteasome system is involved in the pathogenic process of this disease. In our previous study, neither iron accumulation in the brain nor motor and behavioral dysfunctions were noted in Cp−/− mice [17]. This prompted us to treat Cp−/− mice
K. Kaneko et al. / Neuroscience Letters 446 (2008) 56–58
Fig. 1. Immunoblot of brain homogenate using a rabbit anti-Cp polyclonal antibody. Lane M: molecular weight marker, lane 1: Cp−/−, R− mouse, lane 2: Cp−/−, R+ mouse, lane 3: Cp+/+, R− mouse, lane 4: Cp+/+, R+ mouse. Cp+/+ mice contain large amounts of the 132 kDa full-length Cp in the brain, whereas Cp−/− mice lack Cp.
with rotenone. The aim of this study was to elucidate the neuroprotective effects of Cp under the neurotoxic conditions induced by rotenone. The derivation of the Cp-deficient (Cp−/−) mouse used in this study has been already described [17]. The genetic background of these mice was converted from BALB/c to C57BL/10 by seven successive congenic matings of Cp+/− male mice with wild-type C57BL/10 female mice. All animal procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and they were approved by the Institutional Animal Care and Use Committee of the Shinshu University. All appropriate measures were taken to minimize pain and discomfort in the experimental animals. 13 week-old mice were treated with 10 mg/(kg day) rotenone for 4 weeks using an osmotic pump (Alzet) subcutaneously implanted in the lower back. The rotenone was first dissolved in dimethyl sulfoxide (DMSO, Wako, Japan), then the solution was mixed with peanuts oil in the same volume of DMSO. The rotenone-untreated mice were given a same osmotic pump poured into DMSO and peanuts oil alone. As spontaneous neurodegeneration might occur in aged (over 24 months) mice brains, as shown in the study of Jeong et al. [5], we used young (13-weekold) male mice to minimize the influence of aging. To elucidate the effect of Cp under a rotenone-induced neurotoxic conditions, we established the following four types of mice: (1) rotenone-treated, Cp-deficient mice (Cp−/−R+), (2) rotenone-untreated, Cp-deficient mice (Cp−/−R−), (3) rotenone-treated, wild-type mice (Cp+/+R+), and (4) rotenone-untreated, wild-type mice (Cp+/+R−). We prepared three mice in each condition. After 4 weeks of treatment with rotenone, the mice were euthanized and their whole brains were removed. One hemisphere was weighed and homogenized in 3 ml/g of high salt buffer (50 mM Tris–HCl, pH 7.5, 750 mM
57
NaCl) supplemented with a cocktail of protease inhibitors (complete, Mini. Roche). The samples were centrifuged at 10,000 × g for 20 min. Supernatants were analyzed to confirm Cp deficiency using immunoblotting as described previously [17]. The remaining hemisphere of the removed brain was formalin fixed. After sufficient fixation, the brain was paraffin embedded and cut at 5 m. The sections were deparaffinized, and hydrated through graded alcohols. These sections were first microwaved in 0.1 M sodium citrate, pH 6.5. They were then incubated with 3% aqueous hydrogen peroxide (H2 O2 ) (to remove endogenous peroxidase activity), and in 10% goat serum in phosphate buffered saline for 1 h. Subsequently, sections were incubated overnight at 4 ◦ C with one of the following primary antibodies (dilutions in parentheses): rabbit anti-ubiquitin (1:500, DAKO) or mouse anti-acrolein (1:100, Nippon yushi, Tokyo, Japan). These incubations were followed by an incubation period with biotinylated anti-rabbit or anti-mouse IgG secondary antibody (1:300, DAKO), streptavidin–HRP conjugate (1:300, GIBCO BRL), and reacted with diaminobenzidine (0.3 mg/ml, SIGMA) in 50 mM Tris buffer (pH 7.4) with 0.001% H2 O2 . Finally, the sections were lightly counterstained with hematoxylin. The absence of the Cp protein was reconfirmed in the brain homogenate in Cp−/− mice with immunoblotting (Fig. 1). Antiacrolein antibody reacted strongly to hippocampal neuronal cytoplasm in Cp−/−R+ mice. Very weak staining was observed in the other three types of mice. There was no clear difference in the immunoreactivity between these three types of mice (Fig. 2). The same results were seen in another regions, such as the cerebellum, caudoputamen and thalamus, but the lesion was most striking in the hippocampus. Similar results were obtained with immunohistochemistry using an anti-ubiquitin antibody (Fig. 3). Contrast to the prominent lesions in the brain with aceruloplasminemia, neuronal cells were more severely affected than glial cells in Cp−/−, R+ mice brain. Cp−/−R+ mice had a tendency to exhibit reduced activity or to stagger, although we did not evaluate motor functions or behaviors precisely. In the present study, we showed that the immunoreactivity of acrolein and ubiquitin were enhanced in the brains of Cp−/−R+ mice. These findings mimicked the pathological processes in the brains of aceruloplasminemia patients. Thus, rotenone treatment on a Cp−/− mouse will be useful as an animal model for aceruloplasminemia. It may explain the reason why the activity of the mitochondrial respiratory chain complex I was markedly reduced in the brains of aceruloplasminemia patients [10].
Fig. 2. Immunostaining of the hippocampus of rotenone-treated/untreated mice with an anti-acrolein antibody. (A) Cp−/−, rotenone-treated mouse; (B) Cp−/−, otenoneuntreated mouse; (C) Cp+/+, rotenone-treated mouse; (D) Cp+/+, rotenone-untreated mouse. Hippocampal cells of Cp−/−, rotenone-treated mouse show a strong immunoreactivity with acrolein (A). Scale bars: 10 m.
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K. Kaneko et al. / Neuroscience Letters 446 (2008) 56–58
Fig. 3. Immunostaining for ubiquitin in the hippocampi of mice. (A) Cp−/−, rotenone-treated mouse; (B) Cp−/−, rotenone-untreated mouse; (C) Cp+/+, rotenone-treated mouse; (D) Cp+/+, rotenone-untreated mouse. Numerous cells in the hippocampal pyramidal layer of Cp−/−, rotenone-treated mouse are strongly positive for ubiquitin (A). Weakly positive cells are seen in the same region of the other conditions (B–D). Insets are high-power magnification of the boxed area in each panel. Scale bars: 500 m; inset: 50 m.
In conclusion, lipid peroxidation and ubiquitination were enhanced in Cp−/−R+ mice. This indicates that Cp plays a protective role against rotenone-induced neurotoxicity. References [1] R. Betarbet, T.B. Sherer, G. MacKenzie, M. Garcia-Osuna, A.V. Panov, J.T. Greenamyre, Chronic systemic pesticide exposure reproduces features of Parkinson’s disease, Nat. Neurosci. 3 (2000) 1301–1306. [2] V.C. Culotta, J.D. Gitlin, Disorders of copper transport, in: C.R. Scriver, A.L. Beaudet, W.S. Sly, D. Valle (Eds.), The Metabolic and Molecular Bases of Inherited Disease, vol. II, McGraw Hill, New York, 2001, pp. 3105–3126. [3] B. Halliwell, J.M.C. Gutteridge, Free Radicals in Biology and Medicine, 3rd ed., Oxford University Press, London, 1999. [4] P. Jenner, Oxidative stress in Parkinson’s disease, Ann. Neurol. 53 (Suppl. 3) (2003) s26–s38. [5] S.Y. Jeong, S. David, Age-related changes in iron homeostasis and cell death in the cerebellum of ceruloplasmin-deficient mice, J. Neurosci. 26 (2006) 9810–9819. [6] K. Kaneko, A. Nakamura, K. Yoshida, F. Kametani, K. Higuchi, S. Ikeda, Glial fibrillary acidic protein is greatly modified by oxidative stress in aceruloplasminemia brain, Free Radic. Res. 36 (2002) 303–306. [7] K. Kaneko, K. Yoshida, K. Arima, S. Ohara, H. Miyajima, T. Kato, M. Ohta, S. Ikeda, Astrocytic deformity and globular structures are characteristic of the brains of patients with aceruloplasminemia, J. Neuropathol. Exp. Neurol. 61 (2002) 1069–1077. [8] T. Kawanami, T. Kato, M. Daimon, M. Tominaga, H. Sasaki, K. Maeda, S Arai, Y. Shikama, T. Katagiri, Hereditary caeruloplasmin deficiency: clinicopathological study of a patient, J. Neurol. Neurosurg. Psychiatry 61 (1996) 506–509. [9] J. Lowe, J. Mayer, M. Landon, R. Layfield, Ubiquitin and the molecular pathology of neurodegenerative disease, Adv. Exp. Med. Biol. 487 (2001) 169–186.
[10] H. Miyajima, S. Kono, Y. Takahashi, M. Sugimoto, Increased lipid peroxidation and mitochondrial dysfunction in aceruloplasminemia brains, Blood Cells Mol. Dis. 29 (2002) 433–438. [11] H. Miyajima, Y. Nishimura, K. Mizoguchi, M. Sakamoto, T. Shimizu, N. Honda, Familial apoceruloplasmin deficiency associated with blepharospasm and retinal degeneration, Neurology 37 (1987) 761–767. [12] H. Morita, S. Ikeda, K. Yamamoto, S. Morita, K. Yoshida, S. Nomoto, M. Kato, N. Yanagisawa, Hereditary ceruloplasmin deficiency with hemosiderosis: a clinicopathological study of a Japanese family, Ann. Neurol. 37 (1995) 646–656. [13] S. Osaki, D.A. Johnson, E. Frieden, The possible significance of the ferrous oxidase activity of ceruloplasmin in normal human serum, J. Biol. Chem. 241 (1966) 2746–2751. [14] B.N. Patel, S. David, A novel glycosylphosphatidylinositol-anchored form of ceruloplasmin is expressed by mammalian astrocytes, J. Biol. Chem. 272 (1997) 20185–20190. [15] S. Raha, B.H. Robinson, Mitochondria, oxygen free radicals, disease and aging, Trends Biochem. Sci. 25 (2000) 502–508. [16] F. Richter, M. Hamann, A. Richter, Chronic rotenone treatment induces behavioral effect but no pathological signs of parkinsonism in mice, J. Neurosci. Res. 85 (2007) 681–691. [17] K. Yamamoto, K. Yoshida, Y. Miyagoe, A. Ishikawa, K. Hanaoka, S. Nomoto, K. Kaneko, S. Ikeda, S. Takeda, Quantitative evaluation of expression of ironmetabolism genes in ceruloplasmin-deficient mice, Biochim. Biophys. Acta 1588 (2002) 195–202. [18] K. Yoshida, K. Furihata, S. Takeda, A. Nakamura, K. Yamamoto, H. Morita, S. Hiyamuta, S. Ikeda, N. Shimizu, N. Yanagisawa, A mutation in the ceruloplasmin gene is associated with systemic hemosiderosis in humans, Nat. Genet. 9 (1995) 267–272. [19] K. Yoshida, K. Kaneko, H. Miyajima, T. Tokuda, A. Nakamura, M. Kato, S. Ikeda, Increased lipid peroxidation in the brains of aceruloplasminemia patients, J. Neurol. Sci. 175 (2000) 91–95.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Increased vulnerability to rotenone-induced neurotoxicity in ceruloplasmin-deficient mice Kazuma Kaneko ∗ , Akiyo Hineno, Kunihiro Yoshida, Shu-ichi Ikeda Department of Neurology and Rheumatology, Shinshu University School of Medicine, 3-1-1 Asahi, 390-8621 Matsumoto, Japan
a r t i c l e
i n f o
Article history: Received 14 July 2008 Received in revised form 15 August 2008 Accepted 30 August 2008 Keywords: Ceruloplasmin Aceruloplasminemia Rotenone Ubiquitin Acrolein Oxidative stress
a b s t r a c t Ceruloplasmin (Cp) is the strongest ferroxidase in human plasma. Hereditary deficiency of this protein, named aceruloplasminemia, is an interesting model to elucidate the pathogenesis and pathophysiology of neurodegeneration induced by oxidative stress. Enhanced oxidative stress due to excessive iron accumulation is observed in the brains of aceruloplasminemia patients. Rotenone, a selective mitochondrial complex I inhibitor, induces neurodegeneration mimicking Parkinson’s disease. We investigated the influence of Cp deficiency upon neurodegeneration using rotenone-treated, Cp-deficient mouse brains. Immunohistochemical examination showed that acrolein, one of the products of lipid peroxides, and ubiquitin were more markedly immunoreacted in the brains of rotenone-treated, Cp-deficient mice than in rotenone-untreated, Cp-deficient or rotenone-treated, wild-type mice. These molecules were localized in neuronal cells. These results suggested that rotenone-induced lipid peroxidation and accumulation of ubiquitin immunoreactivity were enhanced in the absence of Cp. Therefore, Cp may protect neuronal cells from oxidative stress-induced neurodegeneration. © 2008 Elsevier Ireland Ltd. All rights reserved.
Ceruloplasmin (Cp) is a copper-binding ferroxidase that is mainly produced by hepatocytes and secreted into plasma. Cp oxidizes ferrous iron (Fe2+ ) to ferric iron (Fe3+ ) and can facilitate iron loading onto transferrin [3,13]. In the brain, the majority of Cp is derived from astrocytes and is located at the surface of astrocytes as a glycosylphosphatidylinositol (GPI)-anchored form [14]. In the state of Cp deficiency, it is considered that Fe2+ is increased, resulting in an acceleration of hydroxyl radical (HO• ) production in the presence of hydrogen peroxide (H2 O2 ), named the Fenton reaction [3]. Thus, it is likely that oxidative stress is enhanced in Cp-deficient conditions. Aceruloplasminemia, caused by mutations in the Cp gene [2], provides a good model to evaluate the influence of iron-induced oxidative stress upon neurodegeneration. Aceruloplasminemia is characterized clinically by adult-onset diabetes mellitus, retinal degeneration and various neurological symptoms such as involuntary movements, cerebellar ataxia, parkinsonism and dementia [11,18]. The neuropathological findings are summarized as intracytoplasmic iron deposition, and neuronal cell loss [12,8]. Indeed, Jeong and David reported that cerebellar cell loss and iron deposition were increased in aged Cp-deficient mice [5]. Further, we previously reported that the enlarged deformed astrocytes and ubiquitin-positive spheroidal structures are pathological character-
∗ Corresponding author. Tel.: +81 263 37 2673; fax: +81 263 37 3427. E-mail address: [email protected] (K. Kaneko). 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.08.089
istics of the brains of aceruloplasminemia patients [7]. In addition, it has been reported that several markers of oxidative stress, such as malondialdehyde (MDA), 4-hydroxy-2-nonenal (HNE) and carbonylated protein, are significantly elevated in the brains of patients with this disorder [19,6]. These data strongly support the hypothesis that enhanced oxidative stress causes neuronal cell death in aceruloplasminemia. There is evidence that oxidative stress contributes to neuronal dysfunction and death in several neurodegenerative diseases, including Parkinson’s disease [4]. In Parkinson’s disease, reduced activity of the mitochondrial respiratory chain complex I in dopaminergic neurons is one of the factors enhancing oxidative stress [15]. Rotenone, a neurotoxic pesticide, is a selective inhibitor of the mitochondrial respiratory chain complex I, as does 1methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (MPTP). The chronic infusion of rotenone has been shown to induce neurodegeneration or motor dysfunction mimicking parkinsonian features in the rat or mouse [1,16]. Ubiquitin, which has a central role in protein degradation [9], is overexpressed in the brains of these animals, suggesting the accumulation of damaged proteins targeted for proteasomal degradation. The presence of ubiquitin-positive structures in the brains of aceruloplasminemia patients indicates that the ubiquitin–proteasome system is involved in the pathogenic process of this disease. In our previous study, neither iron accumulation in the brain nor motor and behavioral dysfunctions were noted in Cp−/− mice [17]. This prompted us to treat Cp−/− mice
K. Kaneko et al. / Neuroscience Letters 446 (2008) 56–58
Fig. 1. Immunoblot of brain homogenate using a rabbit anti-Cp polyclonal antibody. Lane M: molecular weight marker, lane 1: Cp−/−, R− mouse, lane 2: Cp−/−, R+ mouse, lane 3: Cp+/+, R− mouse, lane 4: Cp+/+, R+ mouse. Cp+/+ mice contain large amounts of the 132 kDa full-length Cp in the brain, whereas Cp−/− mice lack Cp.
with rotenone. The aim of this study was to elucidate the neuroprotective effects of Cp under the neurotoxic conditions induced by rotenone. The derivation of the Cp-deficient (Cp−/−) mouse used in this study has been already described [17]. The genetic background of these mice was converted from BALB/c to C57BL/10 by seven successive congenic matings of Cp+/− male mice with wild-type C57BL/10 female mice. All animal procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and they were approved by the Institutional Animal Care and Use Committee of the Shinshu University. All appropriate measures were taken to minimize pain and discomfort in the experimental animals. 13 week-old mice were treated with 10 mg/(kg day) rotenone for 4 weeks using an osmotic pump (Alzet) subcutaneously implanted in the lower back. The rotenone was first dissolved in dimethyl sulfoxide (DMSO, Wako, Japan), then the solution was mixed with peanuts oil in the same volume of DMSO. The rotenone-untreated mice were given a same osmotic pump poured into DMSO and peanuts oil alone. As spontaneous neurodegeneration might occur in aged (over 24 months) mice brains, as shown in the study of Jeong et al. [5], we used young (13-weekold) male mice to minimize the influence of aging. To elucidate the effect of Cp under a rotenone-induced neurotoxic conditions, we established the following four types of mice: (1) rotenone-treated, Cp-deficient mice (Cp−/−R+), (2) rotenone-untreated, Cp-deficient mice (Cp−/−R−), (3) rotenone-treated, wild-type mice (Cp+/+R+), and (4) rotenone-untreated, wild-type mice (Cp+/+R−). We prepared three mice in each condition. After 4 weeks of treatment with rotenone, the mice were euthanized and their whole brains were removed. One hemisphere was weighed and homogenized in 3 ml/g of high salt buffer (50 mM Tris–HCl, pH 7.5, 750 mM
57
NaCl) supplemented with a cocktail of protease inhibitors (complete, Mini. Roche). The samples were centrifuged at 10,000 × g for 20 min. Supernatants were analyzed to confirm Cp deficiency using immunoblotting as described previously [17]. The remaining hemisphere of the removed brain was formalin fixed. After sufficient fixation, the brain was paraffin embedded and cut at 5 m. The sections were deparaffinized, and hydrated through graded alcohols. These sections were first microwaved in 0.1 M sodium citrate, pH 6.5. They were then incubated with 3% aqueous hydrogen peroxide (H2 O2 ) (to remove endogenous peroxidase activity), and in 10% goat serum in phosphate buffered saline for 1 h. Subsequently, sections were incubated overnight at 4 ◦ C with one of the following primary antibodies (dilutions in parentheses): rabbit anti-ubiquitin (1:500, DAKO) or mouse anti-acrolein (1:100, Nippon yushi, Tokyo, Japan). These incubations were followed by an incubation period with biotinylated anti-rabbit or anti-mouse IgG secondary antibody (1:300, DAKO), streptavidin–HRP conjugate (1:300, GIBCO BRL), and reacted with diaminobenzidine (0.3 mg/ml, SIGMA) in 50 mM Tris buffer (pH 7.4) with 0.001% H2 O2 . Finally, the sections were lightly counterstained with hematoxylin. The absence of the Cp protein was reconfirmed in the brain homogenate in Cp−/− mice with immunoblotting (Fig. 1). Antiacrolein antibody reacted strongly to hippocampal neuronal cytoplasm in Cp−/−R+ mice. Very weak staining was observed in the other three types of mice. There was no clear difference in the immunoreactivity between these three types of mice (Fig. 2). The same results were seen in another regions, such as the cerebellum, caudoputamen and thalamus, but the lesion was most striking in the hippocampus. Similar results were obtained with immunohistochemistry using an anti-ubiquitin antibody (Fig. 3). Contrast to the prominent lesions in the brain with aceruloplasminemia, neuronal cells were more severely affected than glial cells in Cp−/−, R+ mice brain. Cp−/−R+ mice had a tendency to exhibit reduced activity or to stagger, although we did not evaluate motor functions or behaviors precisely. In the present study, we showed that the immunoreactivity of acrolein and ubiquitin were enhanced in the brains of Cp−/−R+ mice. These findings mimicked the pathological processes in the brains of aceruloplasminemia patients. Thus, rotenone treatment on a Cp−/− mouse will be useful as an animal model for aceruloplasminemia. It may explain the reason why the activity of the mitochondrial respiratory chain complex I was markedly reduced in the brains of aceruloplasminemia patients [10].
Fig. 2. Immunostaining of the hippocampus of rotenone-treated/untreated mice with an anti-acrolein antibody. (A) Cp−/−, rotenone-treated mouse; (B) Cp−/−, otenoneuntreated mouse; (C) Cp+/+, rotenone-treated mouse; (D) Cp+/+, rotenone-untreated mouse. Hippocampal cells of Cp−/−, rotenone-treated mouse show a strong immunoreactivity with acrolein (A). Scale bars: 10 m.
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K. Kaneko et al. / Neuroscience Letters 446 (2008) 56–58
Fig. 3. Immunostaining for ubiquitin in the hippocampi of mice. (A) Cp−/−, rotenone-treated mouse; (B) Cp−/−, rotenone-untreated mouse; (C) Cp+/+, rotenone-treated mouse; (D) Cp+/+, rotenone-untreated mouse. Numerous cells in the hippocampal pyramidal layer of Cp−/−, rotenone-treated mouse are strongly positive for ubiquitin (A). Weakly positive cells are seen in the same region of the other conditions (B–D). Insets are high-power magnification of the boxed area in each panel. Scale bars: 500 m; inset: 50 m.
In conclusion, lipid peroxidation and ubiquitination were enhanced in Cp−/−R+ mice. This indicates that Cp plays a protective role against rotenone-induced neurotoxicity. References [1] R. Betarbet, T.B. Sherer, G. MacKenzie, M. Garcia-Osuna, A.V. Panov, J.T. Greenamyre, Chronic systemic pesticide exposure reproduces features of Parkinson’s disease, Nat. Neurosci. 3 (2000) 1301–1306. [2] V.C. Culotta, J.D. Gitlin, Disorders of copper transport, in: C.R. Scriver, A.L. Beaudet, W.S. Sly, D. Valle (Eds.), The Metabolic and Molecular Bases of Inherited Disease, vol. II, McGraw Hill, New York, 2001, pp. 3105–3126. [3] B. Halliwell, J.M.C. Gutteridge, Free Radicals in Biology and Medicine, 3rd ed., Oxford University Press, London, 1999. [4] P. Jenner, Oxidative stress in Parkinson’s disease, Ann. Neurol. 53 (Suppl. 3) (2003) s26–s38. [5] S.Y. Jeong, S. David, Age-related changes in iron homeostasis and cell death in the cerebellum of ceruloplasmin-deficient mice, J. Neurosci. 26 (2006) 9810–9819. [6] K. Kaneko, A. Nakamura, K. Yoshida, F. Kametani, K. Higuchi, S. Ikeda, Glial fibrillary acidic protein is greatly modified by oxidative stress in aceruloplasminemia brain, Free Radic. Res. 36 (2002) 303–306. [7] K. Kaneko, K. Yoshida, K. Arima, S. Ohara, H. Miyajima, T. Kato, M. Ohta, S. Ikeda, Astrocytic deformity and globular structures are characteristic of the brains of patients with aceruloplasminemia, J. Neuropathol. Exp. Neurol. 61 (2002) 1069–1077. [8] T. Kawanami, T. Kato, M. Daimon, M. Tominaga, H. Sasaki, K. Maeda, S Arai, Y. Shikama, T. Katagiri, Hereditary caeruloplasmin deficiency: clinicopathological study of a patient, J. Neurol. Neurosurg. Psychiatry 61 (1996) 506–509. [9] J. Lowe, J. Mayer, M. Landon, R. Layfield, Ubiquitin and the molecular pathology of neurodegenerative disease, Adv. Exp. Med. Biol. 487 (2001) 169–186.
[10] H. Miyajima, S. Kono, Y. Takahashi, M. Sugimoto, Increased lipid peroxidation and mitochondrial dysfunction in aceruloplasminemia brains, Blood Cells Mol. Dis. 29 (2002) 433–438. [11] H. Miyajima, Y. Nishimura, K. Mizoguchi, M. Sakamoto, T. Shimizu, N. Honda, Familial apoceruloplasmin deficiency associated with blepharospasm and retinal degeneration, Neurology 37 (1987) 761–767. [12] H. Morita, S. Ikeda, K. Yamamoto, S. Morita, K. Yoshida, S. Nomoto, M. Kato, N. Yanagisawa, Hereditary ceruloplasmin deficiency with hemosiderosis: a clinicopathological study of a Japanese family, Ann. Neurol. 37 (1995) 646–656. [13] S. Osaki, D.A. Johnson, E. Frieden, The possible significance of the ferrous oxidase activity of ceruloplasmin in normal human serum, J. Biol. Chem. 241 (1966) 2746–2751. [14] B.N. Patel, S. David, A novel glycosylphosphatidylinositol-anchored form of ceruloplasmin is expressed by mammalian astrocytes, J. Biol. Chem. 272 (1997) 20185–20190. [15] S. Raha, B.H. Robinson, Mitochondria, oxygen free radicals, disease and aging, Trends Biochem. Sci. 25 (2000) 502–508. [16] F. Richter, M. Hamann, A. Richter, Chronic rotenone treatment induces behavioral effect but no pathological signs of parkinsonism in mice, J. Neurosci. Res. 85 (2007) 681–691. [17] K. Yamamoto, K. Yoshida, Y. Miyagoe, A. Ishikawa, K. Hanaoka, S. Nomoto, K. Kaneko, S. Ikeda, S. Takeda, Quantitative evaluation of expression of ironmetabolism genes in ceruloplasmin-deficient mice, Biochim. Biophys. Acta 1588 (2002) 195–202. [18] K. Yoshida, K. Furihata, S. Takeda, A. Nakamura, K. Yamamoto, H. Morita, S. Hiyamuta, S. Ikeda, N. Shimizu, N. Yanagisawa, A mutation in the ceruloplasmin gene is associated with systemic hemosiderosis in humans, Nat. Genet. 9 (1995) 267–272. [19] K. Yoshida, K. Kaneko, H. Miyajima, T. Tokuda, A. Nakamura, M. Kato, S. Ikeda, Increased lipid peroxidation in the brains of aceruloplasminemia patients, J. Neurol. Sci. 175 (2000) 91–95.