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Altered expression of DJ-1 in the hippocampal cells following in vivo and in vitro neuronal damage induced by trimethyltin
Neuroscience Letters 440 (2008) 232–236
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Altered expression of DJ-1 in the hippocampal cells following in vivo and in vitro neuronal damage induced by trimethyltin Reiko Nagashima a,1 , Chie Sugiyama a,1 , Yuka Gotoh a , Masanori Yoneyama a , Nobuyuki Kuramoto a , Takahiro Taira b , Hiroyoshi Ariga c , Kiyokazu Ogita a,∗ a b c
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotoge-cho, Hirakata, Osaka 573-0101, Japan Department of Molecular Cell Biology, Interdisciplinary Graduate School of Medicine and Engineering, Yamanashi University, Yamanashi, Japan Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Hokkaido, Japan
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Article history: Received 30 April 2008 Received in revised form 21 May 2008 Accepted 22 May 2008 Keywords: Astrocytes DJ-1 Hippocampus Neuronal death Oxidative stress Trimethyltin
a b s t r a c t Trimethyltin chloride (TMT) is known to produce neuronal damage in the dentate gyrus at least in part via oxidative stress. DJ-1, an oncogene product, is known to act as an anti-oxidant to prevent neuronal damage in dopaminergic neurons. The aim of this study was to determine the alterations in DJ-1 expression in the hippocampal cells of mice after in vivo and in vitro treatment with TMT. In na¨ıve animals, DJ-1 was ubiquitously expressed in the hippocampus, in which the CA1 pyramidal cell layer and dentate granule cell layer had lower and higher levels of it, respectively. An intraperitoneal injection of TMT at the dose of 2.8 mg/kg produced DJ-1 up-regulation in the CA1 pyramidal cell layer, CA3 stratum lucidum, dentate molecular layer, and dentate hilus, but not in the dentate granule cell layer, on day 3–5 post-treatment. Temporary depletion of endogenous glutathione by the prior subcutaneous injection of 2-cyclohexen-1one was effective in facilitating neuronal damage and DJ-1 up-regulation in the dentate gyrus induced by an intraperitoneal injection of TMT at the dose of 2.0 mg/kg. In primary cultures of mouse hippocampal cells, DJ-1 was present in neurons, but not in astrocytes. TMT treatment produced a dramatic expression of DJ-1 in the astrocytes in the cultures. Taken together, our data suggest that the DJ-1 protein is positively regulated in response to oxidative stress induced by TMT. © 2008 Elsevier Ireland Ltd. All rights reserved.
Trimethyltin chloride (TMT) induces neuronal damage in both human and rodent central nervous systems [3,13]. In mice, the damage occurs exclusively in the granule neurons of the dentate gyrus [5,16]. Our recent studies demonstrated the involvement of c-Jun N-terminal kinase/stress-activated protein kinase apoptotic signals [15] in TMT-induced neuronal death in the dentate gyrus. Evidence for the involvement of oxidative stress in TMT neurotoxicity comes from our previous findings that depletion of endogenous glutathione in mice facilitates the dentate granule cell death induced by TMT [24], as well as from other findings that a single injection of TMT into rats produced a rapid increase in the formation of hydroxyl radical and in the levels of malondialdehyde and protein carbonyl [19]. The DJ-1 gene was identified as a novel oncogene [11]. Evidence for the involvement of DJ-1 in Parkinson’s disease comes from the observation that deletion or point mutations of the DJ-1 gene are responsible for the onset of autosomal recessive Parkinsonism (also
∗ Corresponding author. Tel.: +81 72 866 3110; fax: +81 72 866 3110. E-mail address: [email protected] (K. Ogita). 1 These authors contributed equally to this work. 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.05.100
known as PARK7) [1]. Accumulating evidence suggests that DJ1 is a multi-functional protein and plays roles in anti-oxidative stress reactions [2,21,23], transcriptional regulation [12,20], and chaperon reactions [18]. As regards the anti-oxidative reaction, for example, DJ-1 eliminates hydrogen peroxide by oxidizing itself [21]. Knock down of DJ-1 with small interfering RNA results in susceptibility to oxidative stress, endoplasmic reticulum stress, and proteasome inhibition [23]. Furthermore, DJ-1-deficient mice have nigrostriatal dopaminergic dysfunction, motor deficits, and hypersensitivity to neurotoxins such as 1-methyl-4-phenyl-1,2,3,6tetrahydropyrindine (MPTP) and 6-hydroxidopamine [4,7,9]. The objective of the present study was to determine the alterations in DJ-1 expression in the hippocampal cells of mice after in vivo and in vitro treatment with TMT, which causes neuronal death in the dentate gyrus of mice at least in part through oxidative stress. The current in vivo data showed that temporary up-regulation of DJ1 was elicited in particular hippocampal regions, such as the CA1 pyramidal cell layer (pcl), CA3 stratum lucidum (sl), dentate molecular layer (ml), and dentate hilus (hi), following TMT treatment. Further in vitro experiments using hippocampal cultures indicated that DJ-1 was definitely found in astrocytes following neuronal death by TMT, but not in those before TMT treatment.
R. Nagashima et al. / Neuroscience Letters 440 (2008) 232–236
The protocol used here met the guidelines of the Japanese Society for Pharmacology and was approved by the Committee for Ethical Use of Experimental Animals at Setsunan University. Adult male Std-ddY mice, weighing 30–35 g and 5–6 weeks of age, were intraperitoneally injected with TMT (2.8 or 2.0 mg/kg, Wako Pure Chemical Industries Ltd., Osaka, Japan) dissolved in phosphatebuffered saline, and then returned to their home cages until the time of decapitation. For histological assessments, mice were deeply anesthetized with pentobarbital (250 mg/kg, i.p.) and perfused via the heart with saline, followed by 4% (wt/vol) paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). The brains were quickly removed and further fixed with the same fixative solution at 4 ◦ C overnight. Postfixed brains were embedded in paraffin, cut as coronal sections of 2-m thickness with a microtome. Some sections so obtained were counterstained with 0.1% (wt/vol) hematoxylin and others were subjected to immunostaining analysis using a rabbit antibody against single-stranded DNA (ssDNA, Dako Japan Co. Ltd., Kyoto, Japan) or DJ-1 [11], or to Nissl staining with 0.1% (wt/vol) cresyl violet acetate, as described previously [15,16,24]. Stained sections were viewed with an Olympus U-LH100HG fluorescence microscope, and the number of cells was counted by microscopic observation. Quantitative data were obtained from the average of the positive cells in the hippocampi of right and left cerebral hemisphere on a slice. When non-immune rabbit IgG was utilized under the same experimental condition as a negative control, no stain was observed in any section used for this purpose. Primary cultures of hippocampal cells were prepared from 15day-old embryonic ddY mice. In brief, the dissected hippocampus was incubated for 12 min at room temperature in 0.02% EDTA solution. After the medium had been removed, the cells were suspended by gentle trituration in 1:1 mixture of DMEM and nutrient mixture F-12 (DMEM/F12) (Gibco BRL, MD, USA) medium containing 10% (vol/vol) fetal calf serum and other supplements including 33 mM glucose, 2 mM glutamine, 5 mM HEPES, 0.12% sodium bicarbonate, 100 U/mL penicillin, and 100 g/mL streptomycin. After centrifugation and resuspension in the DMFM/F12 medium, the cells were seeded onto poly-l-lysine-coated dishes and incubated at 37 ◦ C in 95% (vol/vol) air/5% (vol/vol) CO2 . The cells were then maintained in the serum-containing medium until 3 DIV (days in vitro), and subsequently in the same medium lacking serum but supplemented with 50 g/mL transferrin, 500 ng/mL insulin, 1 pM -estradiol, 3 nM triiodothyronine, 20 nM progesterone, 8 ng/mL sodium seleniate, and 100 M putrescine until used. The cultures comprised 78% microtubule-associated protein-2 (MAP-2)-positive cells (neurons), 16% glial fibrillary acidic protein (GFAP)-positive cells (astrocytes), and 6% other cells. For immunostaining, cultured cells were washed with Trisbuffered saline (pH 7.5) containing 0.03% (wt/vol) Tween 20 (TBST) and fixed with 4% (wt/vol) paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4) for 20 min at 4 ◦ C. After having been subsequently blocked with 5% (vol/vol) normal goat serum in TBST for 1 h at room temperature, they were then incubated with a mouse monoclonal antibody against MAP-2 (Chemicon International, Temecula, CA) or GFAP (Sigma Chemicals, St. Louis, MO) and a rabbit polyclonal antibody against DJ-1 [11] at 4 ◦ C overnight. After a wash with TBST, the cells were then reacted for 2 h at room temperature with the appropriate secondary antibody, i.e., anti-mouse IgG antibody conjugated with fluorescein isothiocyanate (Sigma Chemicals, St. Louis, MO) for MAP-2 and GFAP or anti-rabbit IgG antibody conjugated with Texas Red (Molecular Probes, Eugene, OR) for DJ1. Finally, the cells were incubated with Hoechst 33342 (1:1000) for 20 min at room temperature and observed under a fluorescence microscope (U-LH100HG, Olympus, Osaka, Japan). Cells were counted in four different visual fields randomly selected on each
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culture dish. The number of cells was determined as the average of those found in the visual fields. Results were expressed as the mean ± S.E., and the statistical significance of differences was determined by using the two-tailed Student’s t-test or the one-way ANOVA with the Bonferroni/Dunnett post hoc test. To assess neuronal damage in the hippocampus after a single injection of TMT at the dose of 2.8 mg/kg, we performed Nissl staining and immunostaining for ssDNA on the hippocampal sections prepared from mice injected with TMT. A large number of ssDNApositive cells were found evenly distributed in the dentate gyrus of the hippocampus on days 2 and 3 after the treatment. Afterwards, however, on days 14–56, no ssDNA-positive cells were found in any parts of the dentate gyrus of animals injected with TMT [ssDNApositive cells/mm2 post-treatment (n = 6): na¨ıve, 0; day 1, 650 ± 45; day 2, 2520 ± 480; day 3, 1930 ± 250; day 7, 290 ± 35; day 28, 0; day 56, 0]. However, neither the CA1 nor CA3 subfield had any ssDNApositive cells at any time post-TMT treatment. In addition to ssDNA staining, Nissl staining revealed that severe neuronal damage had occurred in the granule cells of the dentate gyrus, but not in the pyramidal cells of CA1 and CA3 subfields of the hippocampus on day 2 post-TMT treatment [intact neurons/0.03 mm2 (control vs. day 2) (n = 4): dentate gyrus, 247 ± 14 vs. 69 ± 9 (p < 0.01); CA1, 103 ± 9 vs. 98 ± 7; CA3, 117 ± 7 vs. 100 ± 12]. Next we determined the regional distribution of DJ-1 protein in the hippocampus of mice. In na¨ıve and saline-treated animals, DJ-1 was ubiquitously present within the hippocampus, with a low level in the pcl of the CA1 subfield and a little higher level in the dentate granule cell layer (gcl, Fig. 1a, “Saline”; Fig. 1b, “Na¨ıve”). On day 3 post-TMT treatment, dramatic elevation of DJ-1 levels was seen in the dentate ml, the hi, and sl of the CA3 subfield (Fig. 1a, lower panels). In addition to these regions, the CA1 pyramidal cell layer also had a higher level of DJ-1 in TMT-treated animals than in saline-treated ones. Nevertheless, little change in the DJ-1 level was observed in the dentate granule cell layer after TMT treatment. Fig. 1b shows the time course of altered expression of DJ-1 in the hippocampus following TMT treatment. Although a little elevation of the DJ-1 level was found in the dentate molecular layer and stratum lucidum of the CA3 subfield on day 2 post-TMT treatment, the largest amount of the elevation was seen in the dentate molecular layer, the dentate hilus, and CA3 stratum lucidum at a periods of 3–5 days after TMT treatment. On day 7 post-treatment, the level of DJ-1 was still remained elevated in the dentate molecular layer and hilus. In TMT-treated animals, however, the DJ-1 expression returned to the na¨ıve level at least day 14 post-treatment (data not shown). To assess the possible involvement of oxidative stress in TMTinduced expression of DJ-1 in the hippocampus, we examined the expression of DJ-1 in the hippocampus of glutathione-depleted model mice, which were prepared by prior treatment with 2cyclohexen-1-one (CHO) [14]. A subcutaneous injection of CHO at the dose of 150 mg/kg led to acute reduction in the total glutathione level in the brain. Particularly, the level of total glutathione in the hippocampus was diminished by more than 50% at a period of 1–8 h post-CHO treatment, with gradual recovery to the normal level within 24 h after treatment [total glutathione (mol/g wet weight) post-CHO treatment (n = 4): na¨ıve, 2.25 ± 0.08; 1 h, 1.06 ± 0.12 (p < 0.01); 2h 0.96 ± 0.19 (p < 0.01); 8 h, 1.25 ± 0.09 (p < 0.01); 24 h,1.95 ± 0.29]. In the hippocampus the CHO treatment also produced a further elevation of the lipid peroxidation induced by TMT used at the dose of 2.0 mg/kg. In this glutathione depletion model, ssDNA-positive cells elicited by TMT at the same dose were markedly increased in number in the dentate gyrus by prior treatment with CHO (Fig. 2, right panels) [ssDNA-positive cells/mm2 (n = 4): TMT alone, 320 ± 45; CHO + TMT, 965 ± 206 (p < 0.01)]. Under the same experimental conditions, as expected,
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Fig. 1. Altered expression of DJ-1 in the hippocampus after TMT treatment in vivo. Animals were given either TMT (2.8 mg/kg, i.p.) or saline and then fixed on day 3 (a) or the indicated time points (b) after injection for preparation of hippocampal coronal sections, which were used for immunostaining for DJ-1. These experiments were repeated at lease four times with similar results. cc, central canal; DG, dentate gyrus; gcl, dentate granule cell layer; hi, dentate hilus; ml, dentate molecular layer; or, oriens layer of the hippocampus; pcl, pyramidal cell layer of the hippocampus; sl, stratum lucidum of the hippocampus; sr, stratum radiatum of the hippocampus. Scale bar = 200 m.
TMT-induced elevation of DJ-1 in the dentate molecular layer and hilus was also enhanced by prior treatment with CHO (Fig. 2, left panels). Next we determined the change in DJ-1 expression in primary cultures of the hippocampal cells following TMT treatment. Immunostaining revealed that DJ-1 was ubiquitously present in the perikarya of intact neurons positive for MAP-2 (Fig. 3a, control) [DJ1-positive cells/MAP-2-positive cells (%, n = 4): control, 99.6 ± 0.3; TMT (48 h), 80.6 ± 10.0]. TMT at 5 M produced almost loss of MAP-2-positive cells on day 2 post-treatment (Fig. 3a, TMT) [MAP2-positive cells/mm2 (n = 4): control, 469 ± 84; TMT (48 h), 13 ± 3
(p < 0.01)]. However, DJ-1 was still present in MAP-2-negative cells (damaged neurons). Unlike neurons, astrocytes were resistant to TMT at the concentration of 5 M [GFAP-positive cells/mm2 (n = 4): control, 71 ± 9; TMT (48 h), 69 ± 14]. Although few GFAP-positive cells had DJ-1 under non-treatment conditions, surprisingly, dramatically enhanced expression of DJ-1 was seen in the perikarya of GFAP-positive cells post-TMT treatment (Fig. 3b) [DJ-1-positive cells/GFAP-positive cells (%) (n = 4): control, 6.3 ± 3.3; TMT (48 h), 98.4 ± 1.6 (p < 0.01)]. A large body of evidence suggests a key role of oxidative stress in neurodegenerative diseases such as Alzheimer’s disease, Parkin-
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Fig. 2. Enhancement of TMT-induced expression of DJ-1 by glutathione depletion in the dentate gyrus in vivo. Animals were given TMT (2.0 mg/kg, i.p.) 1 h after injection of CHO (150 mg/kg, s.c.) and then fixed on day 3 after treatment. Subsequently, hippocampal coronal sections were prepared for immunostaining for DJ-1 and ssDNA. These experiments were repeated at lease four times with similar results. Scale bar = 100 m.
son’s disease, and amyotrophic lateral sclerosis. DJ-1 is known to act as a versatile pro-survival factor, such as being an anti-oxidative factor and a molecular chaperone, in dopaminergic neurons of the substantia nigra pars compacta [22]. In the present study, we demonstrated for the first time a marked up-regulation of DJ-1 in the dentate molecular layer, hilus, CA1 pyramidal cells, and CA3 stratum lucidum post-TMT treatment. Further, the findings that temporary depletion of endogenous glutathione produced an elevation of the DJ-1 level as well as of neuronal damage (ssDNA expression) in the TMT-treated animals led us to propose that DJ-1 was up-regulated in response to oxidative stress induced by
TMT, since glutathione depletion enhances neuronal damage that is caused by oxidative stress in TMT-treated animals. Interestingly, DJ-1 was present at a lower level in the CA1 pyramidal cells than in the CA3 ones and dentate granule cells in na¨ıve animals. This may be a hint to explain at least in part the vulnerability of the CA1 pyramidal cells to ischemic insults in mice, although it has been reported that the greater oxidative stress and loss of the glutamate transporter GLT-1 function selectively in CA1 astrocytes is central to the well-known delayed death of CA1 neurons after ischemic insults [17]. As regards the regional selectivity in TMT neurotoxicity, our previous report demonstrated that TMT caused
Fig. 3. Differential expression of DJ-1 in the hippocampal primary cultures after TMT treatment in vitro. Hippocampal cells at 6 DIV were exposed to either 5 M TMT or vehicle for 2 days and then double-stained for MAP-2 (green)/DJ-1 (red, a) or GFAP (green)/DJ-1 (red, b). The cells were counterstained with Hoechst 33342 (blue). These experiments carried out at least four times with similar results under the same experimental conditions. Scale bar = 50 m.
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neuronal damage in the dentate granule cells, but not in pyramidal cells of the CA1-3 subfields [15]. This phenomenon may be at least in part explained by the current findings that TMT had the ability to enhance DJ-1 expression in the CA1 pyramidal cells, at the earlier stage post-treatment, but not in the dentate granule cells. Hence, further study needs to be done in order to elucidate the functions of DJ-1 expressed in response to the TMT insult. Our present study has provided the first and surprising data showing that the exposure of primary co-cultures of neurons and astrocytes to TMT produced a dramatic facilitation of DJ-1 expression in astrocytes after a 48-h treatment. At that time, most of the neurons showed damage with morphological changes including disappearance of MAP-2 and nuclear condensation. These findings raise two possible mechanisms underlying TMT-induced expression of DJ-1 in astrocytes. The first is that DJ-1 expression in astrocytes is positively regulated in response to neuronal death induced by TMT. This possibility may be supported by a previous proposition that astrocytes are activated by early signaling mechanisms responsible for subsequent neuronal degeneration [6]. Indeed, in vivo treatment with TMT produces a marked elevation of GFAP level, like that of DJ-1 level, in the dentate gyrus following neuronal death in the dentate granule cell layer [5]. The second possibility is that TMT directly activates astrocytes and subsequently up-regulates DJ-1 expression. This possibility comes from reports that direct exposure of cultured astrocytes to TMT leads to the enhanced expression of various signaling substances, such as GFAP, vimentin, adrenomedullin, interleukin 1␣, and tumor necrosis factor-␣ [8,10]. Although astrocytes are resistant to TMT insult, the drastic enhancement of DJ-1 expression in response to TMT may contribute to the resistance in astrocytes. Herein we demonstrated that both in vivo and in vitro TMT treatment up-regulated the level of DJ-1 protein in the hippocampus. Furthermore, we provided the first data that DJ-1 was highly expressed in astrocytes treated with TMT. These findings may mean that DJ-1 plays some functional role in astrocytes activated in response to TMT. In future studies additional in vitro evaluation of hippocampal astrocytes in culture should help us to elucidate the role of and the mechanism responsible for the TMT-induced expression of DJ-1. References [1] V. Bonifati, P. Rizzu, M.J. van Baren, O. Schaap, G.J. Breedveld, E. Krieger, M.C. Dekker, F. Squitieri, P. Ibanez, M. Joosse, J.W. van Dongen, N. Vanacore, J.C. van Swieten, A. Brice, G. Meco, C.M. van Duijn, B.A. Oostra, P. Heutink, Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism, Science 299 (2003) 256–259. ´ M.A. Wilson, D.W. Miller, R. Ahmad, C. McLendon, S. Bandy[2] R.M. Canet-Aviles, opadhyay, M.J. Baptista, D. Ringe, G.A. Petsko, M.R. Cookson, The Parkinson’s disease protein DJ-1 is neuroprotective due to cysteine-sulfinic acid-driven mitochondrial localization, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 9103–9108. [3] L.W. Chang, R.S. Dyer, Early effects of trimethyltin on the dentate gyrus basket cells: a morphological study, J. Toxicol. Environ. Health. 16 (1985) 641–653. [4] L. Chen, B. Cagniard, T. Mathews, S. Jones, H.C. Koh, Y. Ding, P.M. Carvey, Z. Ling, U.J. Kang, X. Zhuang, Age-dependent motor deficits and dopaminergic dysfunction in DJ-1 null mice, J. Biol. Chem. 280 (2005) 21418–21426.
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Altered expression of DJ-1 in the hippocampal cells following in vivo and in vitro neuronal damage induced by trimethyltin Reiko Nagashima a,1 , Chie Sugiyama a,1 , Yuka Gotoh a , Masanori Yoneyama a , Nobuyuki Kuramoto a , Takahiro Taira b , Hiroyoshi Ariga c , Kiyokazu Ogita a,∗ a b c
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotoge-cho, Hirakata, Osaka 573-0101, Japan Department of Molecular Cell Biology, Interdisciplinary Graduate School of Medicine and Engineering, Yamanashi University, Yamanashi, Japan Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Hokkaido, Japan
a r t i c l e
i n f o
Article history: Received 30 April 2008 Received in revised form 21 May 2008 Accepted 22 May 2008 Keywords: Astrocytes DJ-1 Hippocampus Neuronal death Oxidative stress Trimethyltin
a b s t r a c t Trimethyltin chloride (TMT) is known to produce neuronal damage in the dentate gyrus at least in part via oxidative stress. DJ-1, an oncogene product, is known to act as an anti-oxidant to prevent neuronal damage in dopaminergic neurons. The aim of this study was to determine the alterations in DJ-1 expression in the hippocampal cells of mice after in vivo and in vitro treatment with TMT. In na¨ıve animals, DJ-1 was ubiquitously expressed in the hippocampus, in which the CA1 pyramidal cell layer and dentate granule cell layer had lower and higher levels of it, respectively. An intraperitoneal injection of TMT at the dose of 2.8 mg/kg produced DJ-1 up-regulation in the CA1 pyramidal cell layer, CA3 stratum lucidum, dentate molecular layer, and dentate hilus, but not in the dentate granule cell layer, on day 3–5 post-treatment. Temporary depletion of endogenous glutathione by the prior subcutaneous injection of 2-cyclohexen-1one was effective in facilitating neuronal damage and DJ-1 up-regulation in the dentate gyrus induced by an intraperitoneal injection of TMT at the dose of 2.0 mg/kg. In primary cultures of mouse hippocampal cells, DJ-1 was present in neurons, but not in astrocytes. TMT treatment produced a dramatic expression of DJ-1 in the astrocytes in the cultures. Taken together, our data suggest that the DJ-1 protein is positively regulated in response to oxidative stress induced by TMT. © 2008 Elsevier Ireland Ltd. All rights reserved.
Trimethyltin chloride (TMT) induces neuronal damage in both human and rodent central nervous systems [3,13]. In mice, the damage occurs exclusively in the granule neurons of the dentate gyrus [5,16]. Our recent studies demonstrated the involvement of c-Jun N-terminal kinase/stress-activated protein kinase apoptotic signals [15] in TMT-induced neuronal death in the dentate gyrus. Evidence for the involvement of oxidative stress in TMT neurotoxicity comes from our previous findings that depletion of endogenous glutathione in mice facilitates the dentate granule cell death induced by TMT [24], as well as from other findings that a single injection of TMT into rats produced a rapid increase in the formation of hydroxyl radical and in the levels of malondialdehyde and protein carbonyl [19]. The DJ-1 gene was identified as a novel oncogene [11]. Evidence for the involvement of DJ-1 in Parkinson’s disease comes from the observation that deletion or point mutations of the DJ-1 gene are responsible for the onset of autosomal recessive Parkinsonism (also
∗ Corresponding author. Tel.: +81 72 866 3110; fax: +81 72 866 3110. E-mail address: [email protected] (K. Ogita). 1 These authors contributed equally to this work. 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.05.100
known as PARK7) [1]. Accumulating evidence suggests that DJ1 is a multi-functional protein and plays roles in anti-oxidative stress reactions [2,21,23], transcriptional regulation [12,20], and chaperon reactions [18]. As regards the anti-oxidative reaction, for example, DJ-1 eliminates hydrogen peroxide by oxidizing itself [21]. Knock down of DJ-1 with small interfering RNA results in susceptibility to oxidative stress, endoplasmic reticulum stress, and proteasome inhibition [23]. Furthermore, DJ-1-deficient mice have nigrostriatal dopaminergic dysfunction, motor deficits, and hypersensitivity to neurotoxins such as 1-methyl-4-phenyl-1,2,3,6tetrahydropyrindine (MPTP) and 6-hydroxidopamine [4,7,9]. The objective of the present study was to determine the alterations in DJ-1 expression in the hippocampal cells of mice after in vivo and in vitro treatment with TMT, which causes neuronal death in the dentate gyrus of mice at least in part through oxidative stress. The current in vivo data showed that temporary up-regulation of DJ1 was elicited in particular hippocampal regions, such as the CA1 pyramidal cell layer (pcl), CA3 stratum lucidum (sl), dentate molecular layer (ml), and dentate hilus (hi), following TMT treatment. Further in vitro experiments using hippocampal cultures indicated that DJ-1 was definitely found in astrocytes following neuronal death by TMT, but not in those before TMT treatment.
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The protocol used here met the guidelines of the Japanese Society for Pharmacology and was approved by the Committee for Ethical Use of Experimental Animals at Setsunan University. Adult male Std-ddY mice, weighing 30–35 g and 5–6 weeks of age, were intraperitoneally injected with TMT (2.8 or 2.0 mg/kg, Wako Pure Chemical Industries Ltd., Osaka, Japan) dissolved in phosphatebuffered saline, and then returned to their home cages until the time of decapitation. For histological assessments, mice were deeply anesthetized with pentobarbital (250 mg/kg, i.p.) and perfused via the heart with saline, followed by 4% (wt/vol) paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). The brains were quickly removed and further fixed with the same fixative solution at 4 ◦ C overnight. Postfixed brains were embedded in paraffin, cut as coronal sections of 2-m thickness with a microtome. Some sections so obtained were counterstained with 0.1% (wt/vol) hematoxylin and others were subjected to immunostaining analysis using a rabbit antibody against single-stranded DNA (ssDNA, Dako Japan Co. Ltd., Kyoto, Japan) or DJ-1 [11], or to Nissl staining with 0.1% (wt/vol) cresyl violet acetate, as described previously [15,16,24]. Stained sections were viewed with an Olympus U-LH100HG fluorescence microscope, and the number of cells was counted by microscopic observation. Quantitative data were obtained from the average of the positive cells in the hippocampi of right and left cerebral hemisphere on a slice. When non-immune rabbit IgG was utilized under the same experimental condition as a negative control, no stain was observed in any section used for this purpose. Primary cultures of hippocampal cells were prepared from 15day-old embryonic ddY mice. In brief, the dissected hippocampus was incubated for 12 min at room temperature in 0.02% EDTA solution. After the medium had been removed, the cells were suspended by gentle trituration in 1:1 mixture of DMEM and nutrient mixture F-12 (DMEM/F12) (Gibco BRL, MD, USA) medium containing 10% (vol/vol) fetal calf serum and other supplements including 33 mM glucose, 2 mM glutamine, 5 mM HEPES, 0.12% sodium bicarbonate, 100 U/mL penicillin, and 100 g/mL streptomycin. After centrifugation and resuspension in the DMFM/F12 medium, the cells were seeded onto poly-l-lysine-coated dishes and incubated at 37 ◦ C in 95% (vol/vol) air/5% (vol/vol) CO2 . The cells were then maintained in the serum-containing medium until 3 DIV (days in vitro), and subsequently in the same medium lacking serum but supplemented with 50 g/mL transferrin, 500 ng/mL insulin, 1 pM -estradiol, 3 nM triiodothyronine, 20 nM progesterone, 8 ng/mL sodium seleniate, and 100 M putrescine until used. The cultures comprised 78% microtubule-associated protein-2 (MAP-2)-positive cells (neurons), 16% glial fibrillary acidic protein (GFAP)-positive cells (astrocytes), and 6% other cells. For immunostaining, cultured cells were washed with Trisbuffered saline (pH 7.5) containing 0.03% (wt/vol) Tween 20 (TBST) and fixed with 4% (wt/vol) paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4) for 20 min at 4 ◦ C. After having been subsequently blocked with 5% (vol/vol) normal goat serum in TBST for 1 h at room temperature, they were then incubated with a mouse monoclonal antibody against MAP-2 (Chemicon International, Temecula, CA) or GFAP (Sigma Chemicals, St. Louis, MO) and a rabbit polyclonal antibody against DJ-1 [11] at 4 ◦ C overnight. After a wash with TBST, the cells were then reacted for 2 h at room temperature with the appropriate secondary antibody, i.e., anti-mouse IgG antibody conjugated with fluorescein isothiocyanate (Sigma Chemicals, St. Louis, MO) for MAP-2 and GFAP or anti-rabbit IgG antibody conjugated with Texas Red (Molecular Probes, Eugene, OR) for DJ1. Finally, the cells were incubated with Hoechst 33342 (1:1000) for 20 min at room temperature and observed under a fluorescence microscope (U-LH100HG, Olympus, Osaka, Japan). Cells were counted in four different visual fields randomly selected on each
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culture dish. The number of cells was determined as the average of those found in the visual fields. Results were expressed as the mean ± S.E., and the statistical significance of differences was determined by using the two-tailed Student’s t-test or the one-way ANOVA with the Bonferroni/Dunnett post hoc test. To assess neuronal damage in the hippocampus after a single injection of TMT at the dose of 2.8 mg/kg, we performed Nissl staining and immunostaining for ssDNA on the hippocampal sections prepared from mice injected with TMT. A large number of ssDNApositive cells were found evenly distributed in the dentate gyrus of the hippocampus on days 2 and 3 after the treatment. Afterwards, however, on days 14–56, no ssDNA-positive cells were found in any parts of the dentate gyrus of animals injected with TMT [ssDNApositive cells/mm2 post-treatment (n = 6): na¨ıve, 0; day 1, 650 ± 45; day 2, 2520 ± 480; day 3, 1930 ± 250; day 7, 290 ± 35; day 28, 0; day 56, 0]. However, neither the CA1 nor CA3 subfield had any ssDNApositive cells at any time post-TMT treatment. In addition to ssDNA staining, Nissl staining revealed that severe neuronal damage had occurred in the granule cells of the dentate gyrus, but not in the pyramidal cells of CA1 and CA3 subfields of the hippocampus on day 2 post-TMT treatment [intact neurons/0.03 mm2 (control vs. day 2) (n = 4): dentate gyrus, 247 ± 14 vs. 69 ± 9 (p < 0.01); CA1, 103 ± 9 vs. 98 ± 7; CA3, 117 ± 7 vs. 100 ± 12]. Next we determined the regional distribution of DJ-1 protein in the hippocampus of mice. In na¨ıve and saline-treated animals, DJ-1 was ubiquitously present within the hippocampus, with a low level in the pcl of the CA1 subfield and a little higher level in the dentate granule cell layer (gcl, Fig. 1a, “Saline”; Fig. 1b, “Na¨ıve”). On day 3 post-TMT treatment, dramatic elevation of DJ-1 levels was seen in the dentate ml, the hi, and sl of the CA3 subfield (Fig. 1a, lower panels). In addition to these regions, the CA1 pyramidal cell layer also had a higher level of DJ-1 in TMT-treated animals than in saline-treated ones. Nevertheless, little change in the DJ-1 level was observed in the dentate granule cell layer after TMT treatment. Fig. 1b shows the time course of altered expression of DJ-1 in the hippocampus following TMT treatment. Although a little elevation of the DJ-1 level was found in the dentate molecular layer and stratum lucidum of the CA3 subfield on day 2 post-TMT treatment, the largest amount of the elevation was seen in the dentate molecular layer, the dentate hilus, and CA3 stratum lucidum at a periods of 3–5 days after TMT treatment. On day 7 post-treatment, the level of DJ-1 was still remained elevated in the dentate molecular layer and hilus. In TMT-treated animals, however, the DJ-1 expression returned to the na¨ıve level at least day 14 post-treatment (data not shown). To assess the possible involvement of oxidative stress in TMTinduced expression of DJ-1 in the hippocampus, we examined the expression of DJ-1 in the hippocampus of glutathione-depleted model mice, which were prepared by prior treatment with 2cyclohexen-1-one (CHO) [14]. A subcutaneous injection of CHO at the dose of 150 mg/kg led to acute reduction in the total glutathione level in the brain. Particularly, the level of total glutathione in the hippocampus was diminished by more than 50% at a period of 1–8 h post-CHO treatment, with gradual recovery to the normal level within 24 h after treatment [total glutathione (mol/g wet weight) post-CHO treatment (n = 4): na¨ıve, 2.25 ± 0.08; 1 h, 1.06 ± 0.12 (p < 0.01); 2h 0.96 ± 0.19 (p < 0.01); 8 h, 1.25 ± 0.09 (p < 0.01); 24 h,1.95 ± 0.29]. In the hippocampus the CHO treatment also produced a further elevation of the lipid peroxidation induced by TMT used at the dose of 2.0 mg/kg. In this glutathione depletion model, ssDNA-positive cells elicited by TMT at the same dose were markedly increased in number in the dentate gyrus by prior treatment with CHO (Fig. 2, right panels) [ssDNA-positive cells/mm2 (n = 4): TMT alone, 320 ± 45; CHO + TMT, 965 ± 206 (p < 0.01)]. Under the same experimental conditions, as expected,
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Fig. 1. Altered expression of DJ-1 in the hippocampus after TMT treatment in vivo. Animals were given either TMT (2.8 mg/kg, i.p.) or saline and then fixed on day 3 (a) or the indicated time points (b) after injection for preparation of hippocampal coronal sections, which were used for immunostaining for DJ-1. These experiments were repeated at lease four times with similar results. cc, central canal; DG, dentate gyrus; gcl, dentate granule cell layer; hi, dentate hilus; ml, dentate molecular layer; or, oriens layer of the hippocampus; pcl, pyramidal cell layer of the hippocampus; sl, stratum lucidum of the hippocampus; sr, stratum radiatum of the hippocampus. Scale bar = 200 m.
TMT-induced elevation of DJ-1 in the dentate molecular layer and hilus was also enhanced by prior treatment with CHO (Fig. 2, left panels). Next we determined the change in DJ-1 expression in primary cultures of the hippocampal cells following TMT treatment. Immunostaining revealed that DJ-1 was ubiquitously present in the perikarya of intact neurons positive for MAP-2 (Fig. 3a, control) [DJ1-positive cells/MAP-2-positive cells (%, n = 4): control, 99.6 ± 0.3; TMT (48 h), 80.6 ± 10.0]. TMT at 5 M produced almost loss of MAP-2-positive cells on day 2 post-treatment (Fig. 3a, TMT) [MAP2-positive cells/mm2 (n = 4): control, 469 ± 84; TMT (48 h), 13 ± 3
(p < 0.01)]. However, DJ-1 was still present in MAP-2-negative cells (damaged neurons). Unlike neurons, astrocytes were resistant to TMT at the concentration of 5 M [GFAP-positive cells/mm2 (n = 4): control, 71 ± 9; TMT (48 h), 69 ± 14]. Although few GFAP-positive cells had DJ-1 under non-treatment conditions, surprisingly, dramatically enhanced expression of DJ-1 was seen in the perikarya of GFAP-positive cells post-TMT treatment (Fig. 3b) [DJ-1-positive cells/GFAP-positive cells (%) (n = 4): control, 6.3 ± 3.3; TMT (48 h), 98.4 ± 1.6 (p < 0.01)]. A large body of evidence suggests a key role of oxidative stress in neurodegenerative diseases such as Alzheimer’s disease, Parkin-
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Fig. 2. Enhancement of TMT-induced expression of DJ-1 by glutathione depletion in the dentate gyrus in vivo. Animals were given TMT (2.0 mg/kg, i.p.) 1 h after injection of CHO (150 mg/kg, s.c.) and then fixed on day 3 after treatment. Subsequently, hippocampal coronal sections were prepared for immunostaining for DJ-1 and ssDNA. These experiments were repeated at lease four times with similar results. Scale bar = 100 m.
son’s disease, and amyotrophic lateral sclerosis. DJ-1 is known to act as a versatile pro-survival factor, such as being an anti-oxidative factor and a molecular chaperone, in dopaminergic neurons of the substantia nigra pars compacta [22]. In the present study, we demonstrated for the first time a marked up-regulation of DJ-1 in the dentate molecular layer, hilus, CA1 pyramidal cells, and CA3 stratum lucidum post-TMT treatment. Further, the findings that temporary depletion of endogenous glutathione produced an elevation of the DJ-1 level as well as of neuronal damage (ssDNA expression) in the TMT-treated animals led us to propose that DJ-1 was up-regulated in response to oxidative stress induced by
TMT, since glutathione depletion enhances neuronal damage that is caused by oxidative stress in TMT-treated animals. Interestingly, DJ-1 was present at a lower level in the CA1 pyramidal cells than in the CA3 ones and dentate granule cells in na¨ıve animals. This may be a hint to explain at least in part the vulnerability of the CA1 pyramidal cells to ischemic insults in mice, although it has been reported that the greater oxidative stress and loss of the glutamate transporter GLT-1 function selectively in CA1 astrocytes is central to the well-known delayed death of CA1 neurons after ischemic insults [17]. As regards the regional selectivity in TMT neurotoxicity, our previous report demonstrated that TMT caused
Fig. 3. Differential expression of DJ-1 in the hippocampal primary cultures after TMT treatment in vitro. Hippocampal cells at 6 DIV were exposed to either 5 M TMT or vehicle for 2 days and then double-stained for MAP-2 (green)/DJ-1 (red, a) or GFAP (green)/DJ-1 (red, b). The cells were counterstained with Hoechst 33342 (blue). These experiments carried out at least four times with similar results under the same experimental conditions. Scale bar = 50 m.
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neuronal damage in the dentate granule cells, but not in pyramidal cells of the CA1-3 subfields [15]. This phenomenon may be at least in part explained by the current findings that TMT had the ability to enhance DJ-1 expression in the CA1 pyramidal cells, at the earlier stage post-treatment, but not in the dentate granule cells. Hence, further study needs to be done in order to elucidate the functions of DJ-1 expressed in response to the TMT insult. Our present study has provided the first and surprising data showing that the exposure of primary co-cultures of neurons and astrocytes to TMT produced a dramatic facilitation of DJ-1 expression in astrocytes after a 48-h treatment. At that time, most of the neurons showed damage with morphological changes including disappearance of MAP-2 and nuclear condensation. These findings raise two possible mechanisms underlying TMT-induced expression of DJ-1 in astrocytes. The first is that DJ-1 expression in astrocytes is positively regulated in response to neuronal death induced by TMT. This possibility may be supported by a previous proposition that astrocytes are activated by early signaling mechanisms responsible for subsequent neuronal degeneration [6]. Indeed, in vivo treatment with TMT produces a marked elevation of GFAP level, like that of DJ-1 level, in the dentate gyrus following neuronal death in the dentate granule cell layer [5]. The second possibility is that TMT directly activates astrocytes and subsequently up-regulates DJ-1 expression. This possibility comes from reports that direct exposure of cultured astrocytes to TMT leads to the enhanced expression of various signaling substances, such as GFAP, vimentin, adrenomedullin, interleukin 1␣, and tumor necrosis factor-␣ [8,10]. Although astrocytes are resistant to TMT insult, the drastic enhancement of DJ-1 expression in response to TMT may contribute to the resistance in astrocytes. Herein we demonstrated that both in vivo and in vitro TMT treatment up-regulated the level of DJ-1 protein in the hippocampus. Furthermore, we provided the first data that DJ-1 was highly expressed in astrocytes treated with TMT. These findings may mean that DJ-1 plays some functional role in astrocytes activated in response to TMT. In future studies additional in vitro evaluation of hippocampal astrocytes in culture should help us to elucidate the role of and the mechanism responsible for the TMT-induced expression of DJ-1. References [1] V. Bonifati, P. Rizzu, M.J. van Baren, O. Schaap, G.J. Breedveld, E. Krieger, M.C. Dekker, F. Squitieri, P. Ibanez, M. Joosse, J.W. van Dongen, N. Vanacore, J.C. van Swieten, A. Brice, G. Meco, C.M. van Duijn, B.A. Oostra, P. Heutink, Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism, Science 299 (2003) 256–259. ´ M.A. Wilson, D.W. Miller, R. Ahmad, C. McLendon, S. Bandy[2] R.M. Canet-Aviles, opadhyay, M.J. Baptista, D. Ringe, G.A. Petsko, M.R. Cookson, The Parkinson’s disease protein DJ-1 is neuroprotective due to cysteine-sulfinic acid-driven mitochondrial localization, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 9103–9108. [3] L.W. Chang, R.S. Dyer, Early effects of trimethyltin on the dentate gyrus basket cells: a morphological study, J. Toxicol. Environ. Health. 16 (1985) 641–653. [4] L. Chen, B. Cagniard, T. Mathews, S. Jones, H.C. Koh, Y. Ding, P.M. Carvey, Z. Ling, U.J. Kang, X. Zhuang, Age-dependent motor deficits and dopaminergic dysfunction in DJ-1 null mice, J. Biol. Chem. 280 (2005) 21418–21426.
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