Altered expression of heat shock protein 110 family members in mouse hippocampal neurons following trimethyltin treatment in vivo and in vitro

Neuropharmacology 55 (2008) 693–703

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Altered expression of heat shock protein 110 family members in mouse hippocampal neurons following trimethyltin treatment in vivo and in vitro Masanori Yoneyama a, Naoko Iwamoto a, Reiko Nagashima a, Chie Sugiyama a, Koichi Kawada a, Nobuyuki Kuramoto a, Makoto Shuto b, Kiyokazu Ogita a, * a b

Department of Pharmacology, Faculty of Pharmaceutical Sciences, Setsunan University, Hirakata, Osaka 573-0101, Japan Department of Medical Pharmacy, Faculty of Pharmaceutical Sciences, Setsunan University, Hirakata, Osaka 573-0101, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 May 2007 Received in revised form 19 April 2008 Accepted 2 June 2008

The heat shock protein (Hsp) 110 family is composed of HSP105, APG-1, and APG-2. As the response of these proteins to neuronal damage is not yet fully understood, in the present study, we assessed their expression in mouse hippocampal neurons following trimethyltin chloride (TMT) treatment in vivo and in vitro. Although each of these three Hsps had a distinct regional distribution within the hippocampus, a low level of all of them was observed in the granule cell layer of the dentate gyrus in naı¨ve animals. TMT was effective in markedly increasing the level of these Hsps in the granule cell layer, at least 16 h to 4 days after the treatment. In the dentate granule cell layer on day 2 after TMT treatment, HSP105 was expressed mainly in the perikarya of NeuN-positive cells (intact neurons); whereas APG-1 and APG-2 were predominantly found in NeuN-negative cells (damaged neurons as evidenced by signs of cell shrinkage and condensation of chromatin). Assessments using primary cultures of mouse hippocampal neurons exposed to TMT revealed that whereas HSP105 was observed in intact neurons rather than in damaged neurons, APG-1 and APG-2 were detected in both damaged neurons and intact neurons. Taken together, our data suggest that APG-1 and APG-2 may play different roles from HSP105 in neurons damaged by TMT. Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: Heat shock proteins HSP105 APG-1 APG-2 Trimethyltin Neuronal damage

1. Introduction In both prokaryotes and eukaryotes, the majority of heat shock proteins (Hsps) are induced by environmental stress conditions and various agents, including heat shock, anoxia, amino acid analogues, heavy metals, and certain inhibitors of mitochondrial respiration (Lindquist and Craig, 1988; Welch, 1992). Hsps in mammalian cells are classified into several families based on their apparent molecular masses and degrees of structural homology. The major Hsps in mammalian cells are small Hsps (25–28 kDa), HSP60, HSP70 (68– 80 kDa), HSP90 (83–99 kDa), and high-molecular-weight Hsps (Hsp110). In mice, Hsp110 family members are HSP110/105 (Yasuda et al., 1995), APG (ATP and peptide-binding protein in germ cells)-1/ Osp (osmotic protein) 94 (Kaneko et al., 1997b; Kojima et al., 1996), and APG-2 (Kaneko et al., 1997a). The apg-2 gene has been cloned from a mouse testis cDNA library by use of apg-1 cDNA as a probe. The transcript level of apg-2 is not affected by heat shock conditions

* Corresponding author. Department of Pharmacology, Faculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotoge-cho, Hirakata, Osaka 573-0101, Japan. Tel./fax: þ81 (0)72 866 3110. E-mail address: [email protected] (K. Ogita). 0028-3908/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2008.06.009

(Kaneko et al., 1997a; Nonoguchi et al., 1999; Okui et al, 2000), whereas apg-1 and hsp110/105 transcripts are inducible under such conditions (Santos et al., 1998). In addition, Xue et al. (1998) have indicated that transient forebrain ischemia is effective in increasing the levels of transcripts of apg-1, apg-2, and hsp110 in the rat cerebral cortex and hippocampus. Their studies have been supported by the further finding that the expression of APG-2 protein is enhanced in both astrocytes and neurons of the hippocampus, after transient forebrain ischemia in rats (Lee et al., 2002). Also, transient forebrain ischemia is known to elicit ischemia-responsive protein 94 kDa, which has been characterized as a novel member of the Hsp110 family (Yagita et al., 1999, 2001). It thus seems likely that ischemic brain injury enhances the expression of Hsp110 family members. However, an earlier study of ours has indicated a decrease in the level of APG-2 in the hippocampus after kainate treatment, which causes neuronal loss in the hippocampal CA1 and CA3 subfields, but not in the dentate gyrus (Ogita et al., 2001). The molecular basis of selective vulnerability of specific neuronal populations to different insults has been a key focus of attention in neurology and neuropathology. The organotin trimethyltin chloride (TMT), which had been previously used in fungicides and chemostabilizers, selectively induces neuronal damage in both human and rodent central nervous systems (Balaban et al., 1988).

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The pattern of neurodegeneration caused by a single exposure to TMT is different between rats and mice: whereas in rats TMT damages granule neurons of the dentate gyrus and pyramidal cells in CA1 and CA3c/CA4 subfields (Oderfeld-Nowak and Zaremba, 1998), this damage is exclusively restricted to granule neurons of the dentate gyrus in mice (Fiedorowicz et al., 2001). Our previous studies using mice also demonstrated that the c-Jun N-terminal kinase signaling cascade is involved in TMT-induced granule cell death in the dentate gyrus (Ogita et al., 2004) and that TMT treatment markedly leads to enhanced neurogenesis in the dentate gyrus (Ogita et al., 2005). In contrast to the case of TMT, a variety of other insults including kainate and brain ischemia induce neuronal damage in pyramidal cells in CA1 and CA3 subfields of the hippocampus, but not in granule cells of the dentate gyrus, independently of the species used. These previous findings lend support to the proposal that the TMT exposure mouse model is very attractive for studies on neuronal degeneration and regeneration in the dentate gyrus. The objective of the present study was to investigate the expression profiles of Hsp110 family member proteins in the hippocampus before and after TMT-induced neuronal damage in vivo and in vitro. Herein we demonstrate that the levels of Hsp110 family member proteins, i.e., HSP105, APG-1, and AGP-2, were selectively elevated by TMT in granule cells, but not in glial cells (astrocytes and microglia), of the dentate gyrus. Further evaluation showed that whereas HSP105 was located in intact neurons rather than in damaged neurons, both APG-1 and APG-2 were expressed in a large number of damaged neurons as well as in intact neurons, supporting our proposal that APG-1 and APG-2 may play different roles from HSP105 in neurons damaged by TMT. 2. Materials and methods 2.1. Materials A mouse monoclonal antibody against neuronal nuclear antigen (NeuN) was supplied by Chemicon International (Temecula, CA, USA); and a rabbit polyclonal antibody against single-stranded DNA (ssDNA), by Dako Japan Co., Ltd. (Kyoto, Japan). Rabbit polyclonal antibodies against HSP105, APG-1, and APG-2 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). A mouse monoclonal anti-glial fibrillary acidic protein (GFAP) antibody and goat anti-mouse IgG antibody conjugated with fluorescein isothiocyanate (FITC) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Diaminobenzidine/hydrogen peroxide solution came from Histofine, Ninirei Co. (Tokyo, Japan). Streptavidin-biotin complex peroxidase kit was provided by Nacalai Tesque, Inc. (Kyoto, Japan). Mouse IgG blocking reagent (M.O.M. kit) and fluorescein conjugated Griffonia (bandeiraea) simplicifolia isolectin B4 (GSB4) were from Vector Laboratories Inc. (Burlingame, CA, USA). Texas Red goat anti-rabbit IgG antibody was obtained from Molecular Probes (Eugene, OR, USA). TMT and Protein Assay Rapid kit were supplied by Wako Pure Chemical Industries Ltd. (Osaka, Japan). All other chemicals used were of the highest purity commercially available. 2.2. Drug administration in vivo 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. All efforts were invariably made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to in vivo techniques. Adult male Std-ddY mice, weighing 30–35 g and 5–6 weeks of age, were purchased from a local supplier and housed in metallic breeding cages in a room with a light–dark cycle of 12 h/12 h, a humidity of 55%, and a temperature of 23  C, with free access to food and water for at least 4 days before use. The animals were intraperitoneally injected with TMT (2.8 mg/kg) dissolved in phosphate-buffered saline, and then returned to their home cages until the time of decapitation. 2.3. Preparation of protein samples Brains were quickly removed and immersed in homogenizing buffer (described below) at 2  C, after which the frontal cerebral cortex and hippocampus were dissected according to the procedures described by Glowinski and Iversen (1966). The removed tissues were homogenized in 1 mL of homogenizing buffer composed of 10 mM Tris– HCl buffer (pH 7.5) containing 0.32 M sucrose, 1 mM EDTA, 1 mM EGTA, 5 mM dithiothreitol (DTT), phosphatase inhibitors (10 mM sodium b-glycerophosphate and

1 mM sodium orthovanadate), and 1 mg/mL each of protease inhibitors [(p-amidinophenyl)methanesulfonyl fluoride, benzamidine, leupeptin, and antipain]. A Teflonglass homogenizer was used for homogenization. The homogenates were subsequently centrifuged at 1000  g for 10 min. The resulting supernatant (S1 fraction) was used to prepare mitochondrial, microsomes, and cytosol fractions; whereas the pellet (P1 fraction) was suspended in 1 ml of the homogenizing buffer, after which 10% (wt/vol) Nonidet P-40 was added for a final concentration of 0.5% (wt/vol). The suspension was kept on ice for 5 min, and then again centrifuged at 1000  g for 10 min. The resulting pellet was suspended in the homogenizing buffer and used as the nuclear fraction. The S1 fraction obtained by the first centrifugation (1000  g, 10 min) was centrifuged at 15,000  g for 10 min. The mitochondrial fraction was prepared from the pellet (P2 fraction) by using the method described by Clark and Nicklas (1970) with several modifications (Ogita et al., 2002). The supernatant (S2 fraction) obtained from the S1 fraction was further centrifuged at 100,000  g for 1 h. The resulting pellet and supernatant were pooled as microsomes and cytosol fraction, respectively. All samples were stored at 80  C until used. Immunoblot analysis using antibodies against various marker proteins revealed that the microsomes included at least endoplasmic reticulum, synaptic vesicles, and Golgi. Protein concentrations were measured by the method of Watanabe et al. (1986) using the Protein Assay Rapid kit. 2.4. Immunoblot assay Nuclear extract and cytosolic fraction were individually boiled for 5 min in the presence of 2% (wt/vol) sodium dodecylsulfate (SDS), 5% (vol/vol) 2-mercaptoethanol, 10% (vol/vol) glycerol, and 0.01% (wt/vol) bromophenol blue immediately after preparation and then stored at 80  C until used. Immunoblot assays were carried out as described previously (Azuma et al., 1999). Briefly, an aliquot (20 mg protein) of sample was loaded onto a 10% (wt/vol) polyacrylamide gel, electrophoresed, and transferred to a polyvinylidene fluoride membrane. After having been blocked with 5% (wt/vol) skim milk dissolved in washing buffer [Tris-buffered saline containing 0.05% (wt/vol) Tween 20], the membranes were incubated with the desired primary antibody for 2 h at room temperature, washed three times with washing buffer (5 min each time), and subsequently incubated with horseradish peroxidaseconjugated secondary antibodies for 1 h at room temperature. Proteins reactive with the antibody were detected with the aid of Western Lightning Chemoluminescence Reagent Plus and exposure to X-ray films. 2.5. Histological assessment of the hippocampal slices 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. Post-fixed brains were embedded in paraffin, cut as coronal sections of 2-mm thickness with a microtome, and placed on Matsunami-adhesive silane-coated slide glasses (Matsunami Glass Ind., Ltd., Kyoto, Japan). The paraffin-embedded brain sections were deparaffinized with xylene, rehydrated by immersion in ethanol of graded decreasing concentrations of 100% to 50% (vol/vol), and finally washed with water. Some sections obtained were stained with Hoechst 33342 (0.2 mg/mL) and others were subjected to an immunohistochemical procedure for each protein, as described below. Stained sections were viewed with an Olympus U-LH100HG fluorescence microscope, and the number of cells highly expressing each protein under study was counted by microscopic observation. 2.6. ssDNA immunostaining in hippocampal slices For detection of DNA fragmentation, ssDNA immunoreactivity in coronal sections was determined. Sections were washed with TBST and then incubated with 0.03% (vol/vol) H2O2 in methanol for 5 min. After having been blocked with 5% (vol/ vol) normal goat serum in TBST for 1 h at room temperature, the sections were incubated with an anti-ssDNA antibody (1:1000) at 4  C overnight. After another wash with TBST, these sections were then reacted with biotinylated anti-rabbit IgG antibody (1:200) for 30 min at room temperature and subsequently with the reagents of the streptavidin-biotin complex peroxidase kit for 1 h at room temperature. The peroxidase reaction was visualized with diaminobenzidine/hydrogen peroxide solution. Counter staining was performed by use of 1% (wt/vol) methyl green solution (pH 4.0). 2.7. HSP105, APG-1, and APG-2 immunostaining in the hippocampal slices Sections were first heated in 10 mM sodium citrate buffer (pH 7.0) for 10 min in a microwave oven, washed with TBST, and subsequently incubated with 0.03% (vol/ vol) H2O2 in methanol for 5 min. After blockage with 5% (vol/vol) normal goat serum in TBST for 1 h at room temperature, each section was incubated with a given primary antibody (anti-HAP105, 2 mg/mL; anti-APG-1, 2 mg/mL; anti-APG-2, 2 mg/mL) at 4  C overnight. After having been washed with TBST, the sections were reacted with biotinylated secondary antibody against rabbit IgG (7.5 mg/mL) for 30 min at room temperature and subsequently visualized as described above. For double staining, sections were first heated in 10 mM sodium citrate buffer (pH 7.0) for 10 min in a microwave oven. They were blocked by use of the M.O.M kit

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Fig. 1. Expression of ssDNAþ cells in the dentate gyrus after TMT treatment. Animals were intraperitoneally given TMT at a dose of 2.8 mg/kg and then whole-body fixed with paraformaldehyde at the various time points indicated after the administration. Their brains were then removed and embedded in paraffin. Subsequently, coronal sections of the hippocampus were obtained from the paraffin-embedded brains. ssDNA immunostaining of the sections was carried out to determine DNA fragmentation in neuronal cells. (a) Day 2 after the injection of saline (left panel) or TMT (right panel). Light micrographs with a high magnification are shown in each lower panel. (b) The panels at the left side show typical light micrographs of the dentate gyrus in the coronal sections stained for detection of ssDNA. The number of ssDNAþ cells within the dentate gyrus in the sections is presented in the right graph. Black and gray denote ssDNAþ cells and granule cells counterstained, respectively. Values are the mean  S.E. from four to six separate animals. Scale bar ¼ 200 mm.

for 1 h at room temperature, and then incubated with antibodies against NeuN or GFAP at 4  C overnight. After a wash with TBST and subsequently blockage with 5% (vol/vol) normal goat serum in TBST for 1 h at room temperature, the sections were next incubated with antibody against HSP105, APG-1 or APG-2 (2 mg/mL) at 4  C overnight. After another wash with TBST, they were reacted for 2 h at room temperature with a mixture of Texas Red-conjugated anti-rabbit IgG (10 mg/mL) and FITC-conjugated anti-mouse IgG (3 mg/mL) as secondary antibodies. For double staining with GSB4, which isolectin labels microglia, and antibodies against heat shock proteins, sections were microwaved, and then incubated with FITC-conjugated GSB4 (5 mg/mL) for 2 h at room temperature. After having been washed with TBST and subsequently blocked with 5% normal goat serum in TBST for

1 h at room temperature, the sections were next incubated with antibody against HSP105, APG-1, or APG-2 (2 mg/mL) at 4  C overnight. After another wash with TBST, they were reacted for 2 h at room temperature with Texas Red-conjugated antirabbit IgG (10 mg/mL). 2.8. Cell cultures and immunostaining Primary cultures of hippocampal neurons were prepared from 15-day-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 separated suspended by gentle trituration in a 1:1 mixture of DMEM and

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Fig. 2. Subcellular distribution of HSP105, APG-1, and APG-2 in the frontal cerebral cortex of naı¨ve animals. Animals were decapitated for preparation of nuclear, mitochondrial, microsomes, and cytosol fractions from the frontal cerebral cortex and hippocampus as described in the text. Each fraction was subjected to immunoblot analysis for detection of the levels of HSP105, APG-1, and APG-2. Values are the mean  S.E. from eight independent experiments.

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 mg/ 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. At 2 days in vitro (DIV), the cells were treated with cytosine-b-D-arabinofuranoside at 5 mM for 24 h to avoid the growth of proliferative contaminants such as glial cells. The cells were then maintained in the serumcontaining medium until 6 DIV, and subsequently in the same medium lacking serum but supplemented with 50 mg/mL transferrin, 500 ng/mL insulin, 1 pM bestradiol, 3 nM triiodothyronine, 20 nM progesterone, 8 ng/mL sodium seleniate, and 100 mM putrescine. Microtubule-associated protein 2-positive cells increased in a culture time-dependent manner, representing more than 80% of the cultured cells at 3–12 DIV. For immunostaining, cultured cells were washed with 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 10% (vol/vol) normal goat serum in TBST for 1 h at room temperature, they were reacted overnight at 4  C with adequately diluted primary antibody against HSP105, APG-1 or APG-2 (2 mg/mL). After a wash with TBST, they were reacted with the appropriate secondary antibody, anti-rabbit IgG antibody conjugated with Texas Red (10 mg/mL), for 2 h at room temperature. Finally, the cells were incubated with Hoechst 33342 (5 mg/mL) for 20 min at room temperature and observed under a fluorescence microscope (U-LH100HG, Olympus, Osaka, Japan). 2.9. Data analyses All data were expressed as the mean  S.E., and the statistical significance was determined by the two-tailed Student’s t-test or the one-way ANOVA with Bonferroni/Dunnett post hoc test. Densitometric analysis for quantification of data from any assays was carried out with the aid of Atto Densitograph software (Atto Co. Tokyo, Japan).

To assess neuronal damage in the hippocampus after a single injection of TMT at the above dose, we performed Nissl staining and immunostaining for single-stranded DNA (ssDNA) on the hippocampal sections prepared from mice injected with TMT. On day 2 post TMT treatment, cells immunoreactive toward anti-ssDNA antibody (ssDNAþ cells) were exclusively found in the granule cell layer of the dentate gyrus, but not in the pyramidal cell layers of CA1 and CA3 of the hippocampus (Fig. 1a). Only a few ssDNAþ cells were detected in the dentate gyrus on day 1 post TMT treatment, whereas a large number of ssDNAþ cells were evenly found in the dentate gyrus of the hippocampus on days 2 and 3 after the treatment. Afterwards, however, on days 14–56, no ssDNAþ cells were found in any parts of the dentate gyrus of animals injected with TMT (Fig. 1b). In addition to ssDNA staining, Nissl staining revealed that severe neuronal damage had occurred in the granule cells of the dentate positive gyrus, but not in pyramidal cells of CA1 and CA3 subfields of the hippocampus on day 2 post TMT treatment [neurons/0.03 mm2 (control vs. day 2) (n ¼ 4): dentate gyrus, 247  14 vs. 69  9 (p < 0.01); CA1, 103  9 vs. 98  7; CA3, 117  7 vs. 100  12]. Like in the case of Nissl staining, cells positive for NeuN, which is a marker for mature neurons, were also markedly decreased in the dentate gyrus on days 1–3 after the treatment, with a subsequent return to the control level by day 14 after treatment [NeuNþ cells/0.03 mm2 (n ¼ 4): day 0, 226  28; day 1/2, 206  36; day 1, 156  16 (p < 0.05); day 2, 56  9 (p < 0.001); day 7, 186  39; day 14, 201  30 day 56, 197  15].

3. Results 3.1. Neuronal damage in the dentate gyrus after TMT treatment in vivo Following a single intraperitoneal injection of TMT at a dose of 2.8 mg/kg, about 70% of the mice showed persistent tremor with tail biting within 24 h, with this tremor being sustained for up to 3 days after the injection. The remainder of the animals developed severe tremor with motor paralysis of inferior limbs. Less than 10% of the mice died prior to assessment of the neuronal damage.

3.2. Distribution of Hsp110 family members in the brain of naı¨ve mice To assess the subcellular distribution of the Hsp110 family members in the brain, we analyzed by immunoblotting the levels of HSP105, APG-1, and AGP-2 in nuclear, mitochondrial, microsomal, and cytosol fractions prepared from the frontal cerebral cortex of naı¨ve animals. HSP105, APG-1, and APG-2 were similar to each other in their subcellular distribution in this brain region. The levels were the highest in the cytosol fraction, and progressively

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Fig. 3. Regional distribution of HSP105, APG-1, and APG-2 in the hippocampus of naı¨ve animals. Brains in naı¨ve animals were fixed and removed, and then hippocampal coronal sections were prepared from the paraffin-embedded brains. Subsequently, the sections were subjected to immunohistochemical analysis of HSP105 (a), APG-1 (b), and APG-2 (c) by light microscopy as described in Section 2. Each upper panel shows a typical light micrograph of a hippocampal coronal section. The areas of CA1, CA3, and dentate gyrus subfields on the sections are shown at a higher magnification in each lower panel. Instead of each antibody, non-immune IgG was used as a negative control (d). The left and right panels show light micrographs of the sections with and without counterstaining by hematoxylin, respectively. gcl, dentate granule cell layer; sgz, dentate subgranular zone; hi, dentate hilus; ml, dentate molecular layer; pcl, pyramidal cell layer of the hippocampus; sl, stratum lucidum of the hippocampus; or, oriens layer of the hippocampus; sr, stratum radiatum of the hippocampus.

decreased in the order of mitochondrial, microsomal, and nuclear fractions (Fig. 2). We next determined the regional distribution of the Hsp110 family members in the hippocampus. HSP105 was ubiquitously present within the hippocampus, being clearly found in the perikarya of neurons. Among neuronal cells in CA1, CA3, and dentate

gyrus regions, CA3 pyramidal cells had the highest level of HSP105 (Fig. 3a). Although APG-1 and APG-2 were also ubiquitous within the hippocampus, as in the case of HSP105, the levels of APG-1 and APG-2 were higher in the molecular layer (ml) of the dentate gyrus and stratum lucidum (sl) of the CA3 region than in the pyramidal cell layers (pcl; Fig. 3b,c). Unidentified cells in the dentate

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subgranular zone had a high level of APG-2 (Fig. 3c), but not that of APG-1 or HSP105. 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 (Fig. 3d). 3.3. Changes in the level of Hsp110 family members in the granule cells after TMT treatment in vivo To determine the effects of TMT treatment on the expression of Hsp110 family members in the hippocampus, we carried out immunostaining to detect Hsps at various time points post TMT treatment. On day 2 post TMT treatment, whereas CA1 and CA3 regions had no changes in the level of HSP105, APG-1, and APG-2, interestingly, the dentate granule cells were highly sensitive, showing the elevated expression of these Hsps post TMT treatment (Fig. 4a). For all Hsps, significant elevation was seen between 16 h and day 4 post TMT treatment, when severe neuronal damage was observed in the dentate granule cells had been demonstrated in Fig. 1b. However, this elevated expression of these Hsps was completely eliminated by day 7 post treatment (Fig. 4b). Next, we sought to identify the cells positive for each Hsp in the dentate gyrus on day 2 post TMT treatment. Fig. 5a shows the

results of double labeling for NeuN and each Hsp in the dentate granule cell layer on day 2 post treatment. Hsp105 was found in the perikarya of NeuNþ cells. Interestingly, both APG-1 and APG-2 were highly expressed even in all cells negative for NeuN in addition to being detected in the perikarya of NeuNþ cells. Further double labeling with Hoechst 33342 and antibody against each Hsp in the dentate granule cell layer revealed, as expected, that whereas HSP105 was expressed only in intact cells, both APG-1 and APG-2 were present not only in the perikarya of intact cells but also in damaged cells showing signs of cell shrinkage and condensation of chromatin (Fig. 5b). Being sharply different from the co-localization with NeuN, none of the Hsps were co-localized with GSB4 (which labels microglia) or GFAP (which identifies astrocytes) in the dentate granule cells layer (Fig. 5c). 3.4. Changes in expression of Hsp110 family members in the primary cultures of hippocampal neurons after TMT treatment To confirm TMT-induced changes in the expression of HSP105, APG-1, and APG-2 in hippocampal neurons, we examined the effects of TMT exposure on the expression of these three Hsps in the primary cultures of hippocampal neurons. TMT exposure at 5 mM

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Time after injection of TMT Fig. 4. Increased levels of HSP105, APG-1, and APG-2 in the dentate granule cell layer after TMT treatment. Animals were given TMT (2.8 mg/kg, i.p.) and then fixed on day 2 (a) or at the various time points indicated (b) after the injection of TMT. Subsequently, hippocampal coronal sections were prepared from paraffin-embedded brains. The sections were subjected to immunohistochemical analysis of HSP105, APG-1, and APG-2 by light microscopy as described in Section 2. (a) Left panels show typical light micrographs of the sections. Marked areas denote the dentate granule cell layer (gcl), where expression of these proteins was markedly elevated by TMT. The graph at the right denotes the number of cells highly expressing HSP105, APG-1 or APG-2 in each area (mm2) of the dentate gyrus on day 2 after the injection of saline or TMT. (b) Number of cells highly expressing HSP105, APG-1, and APG-2 is presented in the graph. Values are the mean  S.E. from four separate animals. *p < 0.05, **p < 0.01, significantly different from each control value obtained from saline-treated animals.

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Fig. 5. Identification of cells highly expressing HSP105, APG-1, and APG-2 in the dentate gyrus after TMT treatment. Brains were fixed on day 2 after the injection of TMT (2.8 mg/kg, i.p.) and then hippocampal coronal sections were prepared from paraffin-embedded brains. The sections were subjected to double labeling for each of HSP105, APG-1, and APG-2 as described in Section 2. (a) Double labeling for these Hsps (red, upper panels) and NeuN (green, middle panels) in the dentate granule cells. Merged fluorescence micrographs of each of HSP105, APG-1, and APG-2 with NeuN are denoted in lower panels, where the boxed areas of dentate granular cells are shown at higher magnification. (b) Double labeling for the Hsps (red, upper panels) and Hoechst 33342 (blue, middle panels) in the dentate granule cells. Lower panels denote merged fluorescence micrographs of each of HSP105, APG-1, and APG-2 with Hoechst 33342. Asterisks denote damaged cells with nuclear condensation. (c) Upper panels denote merged fluorescence micrographs of these Hsps (red) with GFAP (green) in the dentate gyrus. Lower panels denote merged fluorescence micrographs of each Hsp (red) with GSI-B4 (green). These experiments carried out at least four times with similar results under the same experimental conditions.

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Fig. 5. (Continued).

led to a marked increase in the number of damaged cells showing signs of cell shrinkage and condensation of chromatin, in a timedependent manner [damaged cells (% of total cells) at various exposure times (n ¼ 4): 0 h, 24  5; 6 h, 33  8; 12 h, 65  6 (p < 0.05); 24 h, 79  9 (p < 0.01)]. Immunostaining revealed that all three Hsps were ubiquitously present in the perikarya of untreated intact neurons (Fig. 6a, control). In TMT-exposed neurons, HSP105 existed predominantly in the intact neurons. Contrary to that of HSP105, the expression of APG-1 and APG-2 was observed in damaged neurons as well as in the intact ones (Fig. 6a, TMT). Fig. 6b shows the number of cells positive for each Hsp in the population of damaged cells at 24 h after TMT treatment. Cells positive for APG-1 and APG-2 were definitely more numerous than those for HSP110 [positive cells (% of damaged cells) (n ¼ 4): HSP110, 9  2; APG-1, 57  9 (p < 0.01); APG-2, 54  9 (p < 0.01)]. 4. Discussion The essential importance of the present findings is that in the hippocampal neurons treated with TMT in vivo and in vitro, HSP105 was highly expressed only in the surviving neurons, whereas both APG-1 or APG-2 were detected in both damaged cells and surviving cells. To date, no observation has been carried out about changes in the expression of HSP110 family members in the hippocampus following TMT treatment, although a previous report indicated the induction of hsp70 mRNA in the hippocampal CA3 subregion of rats treated with TMT (Andersson et al., 1997). In the present study, we demonstrated that HSP105, APG-1, and APG-2 were constitutively present in the hippocampus with only minor differences in distribution. This finding is supported by previous reports (Lee et al., 2002; Hylander et al., 2000). Nevertheless, several lines of evidence indicate that HSP110 family proteins are increased in the hippocampus damaged by an ischemic insult. Transient forebrain ischemia increases the transcription of apg-1 and apg-2 in both cerebral cortex and hippocampus of rats 3–24 h after reperfusion (Xue et al., 1998), although the heat shock condition did not facilitate apg-2 transcription in mouse TAMA26 Sertoli cells or NIH/3T3 fibroblasts (Kaneko et al., 1997a). In addition, transcription of ischemiaresponsive protein 94 (irp94), the amino acid sequence of which is

above 90% identical to that of APG-2 in mouse brain, increased in both pyramidal and granule cell layers of the rat hippocampus after transient brain ischemia and reperfusion (Yagita et al., 1999). These previous reports led us to expect an increase in the APG-1 and APG2 levels in the hippocampal dentate gyrus following TMT treatment, since both transient brain ischemia and TMT treatment lead to neuronal loss in the hippocampus. As expected, TMT treatment dramatically enhanced the expression of these HSP110 family members in the dentate granule neurons damaged selectively. Interestingly, further study revealed that HSP105 was expressed only in the surviving neurons, whereas APG-1 and APG-2 were found not only in surviving neurons but also in the damaged ones. We can offer two possible explanations for these findings. The first is that HPS105 may protect neurons against damage. This is supported by a previous report that PC12 cells over-expressing HSP105a exhibited a strong resistance against damage induced by various forms of stress such as serum deprivation, heat shock, hydrogen peroxide, actinomycin D, and etoposide (Hatayama et al., 2001). Furthermore, HSP105a inhibits heat shock-induced activation of c-Jun N-terminal kinase in PC12 cells. In addition, earlier we obtained evidence that TMT-induced neurotoxicity in the dentate granule cells is at least in part involved in the activation of c-Jun N-terminal kinase (Ogita et al., 2004). These findings raise the possibility that HSP105 protects against TMT-induced neurotoxicity by suppressing the activation of c-Jun N-terminal kinase activation, with the result being that HSP105 existed only in the surviving neurons. A second explanation is that HSP110 may have less stability than APG-1 and AGP-2 in damaged cells. In other words, the level of HSP110 may be regulated by a higher turnover rate than that of APG-1 or APG-2. There is no evidence for interpretation of the finding that APG-1 and APG-2 were highly expressed in the damaged neurons until now. However, useful hints may come from previous findings. For example, in Colon 26 cells (Wang et al., 2000) and FM3A cells (Hatayama et al., 1998), HSP110/105 was shown to associate with heat shock cognate protein 70, which is constitutively expressed as a HSP70 family protein in unstressed cells. These findings raise the possibility that HSP110/105 functions cooperatively with HSP70 family proteins and regulates their functions; this complex may represent a chaperone ‘storage compartment’ that awaits a cellular

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a

HSP105

APG-1

APG-2

Control

* * * * TMT

*

*

* *

*

*

*

*

b Positive cells/Damaged cells (%)

*

*

**

**

APG-1

APG-2

60

40

20

0

HSP105

24 h after TMT exposure Fig. 6. TMT-induced changes in the expression of Hsps in the primary cultures of hippocampal neurons. Hippocampal neurons at 6 DIV were exposed to 5 mM TMT for 2 days and then subjected to immunostaining to detect Hsps. The cells were stained with Hoechst 33342 (blue) and antibodies against each Hsp (red). Asterisks denote damaged cells with nuclear condensation. These experiments carried out at least four times with similar results under the same experimental conditions.

requirement for HSP70. As APG-1 and APG-2 are similar to HSP110/ 105 in term of amino acid sequence, it is possible that APG-1 and APG-2 also interact with HSP70 family proteins and have functions similar to those of HSP110/105. The increase in the levels of these proteins by TMT may suppress the supply of heat shock cognate protein 70 in the storage compartment (heat shock cognate protein 70/APG-1 or APG-2 complex), negatively influencing the neuroprotective function of HSP70 family proteins. Nevertheless, a recent report indicated that APG-2 has a chaperone-like activity similar to that of HSP110/105 and an anti-apoptotic activity in hepatocellular carcinomas (Gotoh et al., 2004). Hence, further study needs to be done in order to elucidate the functions of APG-1 and APG-2 expressed in response to the TMT insult. In the present study, we demonstrated that the HSP110 family members examined failed to co-localize with astrocytes and microglia markers in the dentate gyrus after TMT treatment. However, Lee et al. (2002) indicated that after transient forebrain ischemia, AGP-2 was localized in astrocytes as well as in neurons in

the hippocampal CA1 and CA3 subfields. These paradoxical findings may be derived from distinct types of astrocytes in the case of ischemic and TMT insults. Indeed, we have preliminary data showing that TMT leads to the appearance of nestin/GFAP-positive astrocytes in the dentate gyrus (unpublished data). To data, however, there is no report on the expression of nestin/GFAP-positive astrocytes in the hippocampus after transient forebrain ischemia. Herein we demonstrated that in vivo and in vitro TMT treatment up-regulated the levels of HSP110 family members including HSP105, APG-1, and APG-2, in the hippocampal neurons. Furthermore, we provide the first data that HSP105 was highly expressed only in the surviving neurons, whereas both APG-1 and APG-2 were detected in both damaged cells and surviving cells. These findings may mean that APG-1 and APG-2 play different roles from HSP105 in neurons damaged by TMT. In future studies additional in vitro evaluation of hippocampal neurons in primary culture should help us to elucidate the mechanisms responsible for the TMT-induced increase in the levels of the HSP110 family members.

M. Yoneyama et al. / Neuropharmacology 55 (2008) 693–703

Acknowledgement This work was supported in part by grants-in-aid for scientific research to M.Y. and K.O. from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.

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