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Upregulation of ‘peripheral-type’ benzodiazepine receptors in the globus pallidus in a sub-acute rat model of manganese neurotoxicity
Neuroscience Letters 349 (2003) 13–16 www.elsevier.com/locate/neulet
Upregulation of ‘peripheral-type’ benzodiazepine receptors in the globus pallidus in a sub-acute rat model of manganese neurotoxicity Alan S. Hazella,*, Louise Normandina, Bich Nguyenb, Greg Kennedyc a b
Department of Medicine, Hoˆpital Saint-Luc, University of Montreal, Montreal, Quebec, Canada Department of Pathology, Hoˆpital Saint-Luc, University of Montreal, Montreal, Quebec, Canada c Department of Engineering Physics, E´cole Polytechnique, Montreal, Quebec, Canada Received 5 January 2003; accepted 13 May 2003
Abstract Manganese neurotoxicity (MN) is a neurological disorder characterized by selective neuronal loss in the globus pallidus together with characteristic morphological changes known as Alzheimer type II astrocytosis. In order to understand the underlying mechanisms responsible for these processes, we studied early effects of this metal in a sub-acute rat model. Levels of manganese in the globus pallidus were increased by 81% after 1 day of treatment and elevated by 551% compared to controls after 4 days. In addition, manganese treatment led to a 60% increase in ptbr expression, and a 105% increase in levels of its product, the isoquinoline-carboxamide binding protein, a major constituent of the ‘peripheral-type’ benzodiazepine receptor (PTBR) that is localized to astrocytes, in this brain region after 4 days. These results indicate that PTBRs, and possibly neurosteroids, are an early response to manganese exposure and may play a major role in the pathophysiology of MN. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: ‘Peripheral-type’ benzodiazepine receptor; Manganism; Astrocyte; Neurosteroid; Globus pallidus; Manganese neurotoxicity
Manganese is an essential element with an important role in the normal functioning of several enzymes. However, exposure to this metal can lead to manganese neurotoxicity (MN), in which neuronal loss in the globus pallidus [10] and the development of Alzheimer type II astrocytosis [13] are typical features. Alzheimer type II astrocytes have enlarged, pale nuclei with margination of chromatin and prominent nucleoli. The characteristic neuropathological finding of these abnormal cells suggests an important role for astrocytes in the pathophysiology of MN. Unlike central benzodiazepine receptors, the so-called ‘peripheral-type’ benzodiazepine receptor (PTBR) is expressed in high concentrations on the outer mitochondrial membrane, particularly of astrocytes [2]. Its basic structure consists of a 18 kDa isoquinoline carboxamide-binding protein (IBP), a 32 kDa voltage-sensitive anion channel, and a 30 kDa adenine nucleotide carrier [7]. Treatment of cultured astrocytes with manganese results in an upregulation of binding sites for the PTBR ligand 3H-PK 11195 [3]. * Corresponding author. Department of Medicine, Hoˆpital Saint-Luc (CHUM), 1058 St-Denis Street, Montreal, Quebec H2X 3J4, Canada. Tel.: þ 1-514-890-8310, ext. 35740; fax: þ1-514-412-7314. E-mail address: [email protected] (A.S. Hazell).
Although our understanding of the role of increased PTBRs in the pathogenesis of MN is unclear, PTBRs are an important source of neurosteroid production in the brain [11]. In this, the first of a series of investigations on the pathophysiology of MN, we have studied how manganese influences PTBR levels in a sub-acute rat model of this disorder. Male Sprague –Dawley rats (200 g) were placed in one of the following three groups: (A) manganese-treated group (1 day) (n ¼ 25); rats were administered a single dose of manganese (II) chloride (50 mg/kg body weight, i.p.) and allowed to survive for 24 h; (B) manganese-treated group (4 days) (n ¼ 27); rats were administered manganese (II) chloride (50 mg/kg body weight, i.p.) once per day for 4 days; (C) control group (n ¼ 27); rats were treated with carrier only (0.2 ml saline, i.p.). At the appropriate time, animals were decapitated and the brains removed with dissection of the lateral globus pallidus on dry ice, after which the tissue was stored at 2 80 8C until ready for study. Additional animals were perfusion-fixed as previously described [4] for subsequent neuropathological study. Manganese concentrations in brain samples were determined by instrumental neutron activation analysis. Brain
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00649-9
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A.S. Hazell et al. / Neuroscience Letters 349 (2003) 13–16
tissue samples were irradiated for a pre-determined time with the neutron flux of 5 £ 1011 neutron/cm2 per s in a Slowpoke nuclear reactor. After irradiation and before counting, each sample was transferred into a new plastic acid-rinsed vial so that Mn in the radioactive vial would not be detected. Energy emitted was detected as gamma-rays by a germanium detector and the average Mn concentration estimated. Total RNA was extracted from the globus pallidus using TRI Reagent (MRC Inc., Ohio) according to the manufacturer’s protocol. Putative contaminating DNA was eliminated by adding 100 units of RNase-free DNase I per 50 mg of total RNA at 37 8C for 1 h. Purified RNA was then extracted with phenol, precipitated with ethanol and resuspended in diethylpyrocarbonate-treated water. RNA samples were kept at 2 70 8C until use. Expression of the PTBR gene ptbr was investigated by reverse transcription-polymerase chain reaction (RT-PCR). b-Actin was used as an internal standard to monitor loading variations. Total RNA (1 mg) was mixed with 10 mM Tris – HCl (pH 8.3), 1.5 mM MgCl2, 50 mM KCl, 0.01% (w/v) bovine serum albumin, 100 mM dNTPs, primers at 1 mM each, AMV reverse transcriptase (80 units/ml), Taq DNA polymerase (20 units/ml) and 5 mCi/ml [a-32P]dCTP (3000 Ci/mM), for a total reaction volume of 50 ml. The reactions were initially heated at 50 8C for 15 min followed by PCR at 95 8C for 30 s, 59 8C for 1 min and 72 8C for 1 min. Ptbr and b-actin were amplified for 29 cycles and 15 cycles, respectively. Amplification efficiency conditions were determined after a kinetic study to ensure that the PCR product yields were linear with respect to the input RNA. After amplification, the samples were electrophoresed onto 8% polyacrylamide gels, dried, and autoradiographed at 2 70 8C with an intensifying screen. Each band was subsequently excised and Cerenkov radiation was quantitated using a bcounter. Oligonucleotide primers (Sheldon Biotechnology Center, McGill University, Quebec) were designed using the PRIME program (Genetic Computer Group, Wisconsin) based on the following GenBank accession numbers: ptbr, J05122 [16]; b-actin, V01217 [9]. The forward and reverse primer sequences were as follows: 50 -CCATGCTCAACTACTATGTATGGC-30 and 50 -GTACAACTGTCCCCGCATG-30 ( ptbr, 232 bp); 50 -CATCCCCCAAAGTTCTAC30 and 50 -CAAAGCCTTCATACATC-30 (b-actin, 347 bp). The monospecificity of oligonucleotide primers was verified using the nucleotide –nucleotide ‘blastn’ subprogram within BLAST (National Center for Biotechnology Information, Bethesda, MD). For immunoblotting, the lateral globus pallidus was extracted in buffer containing 50 mM Tris, 150 mM NaCl, 2% sodium dodecyl sulfate (SDS), 1% NP-40, 0.5% sodium deoxycholate (pH 8.0) and a protease inhibitor cocktail (Sigma, St. Louis, MO), and centrifuged at 10,000 £ g for 10 min at 4 8C. Aliquots of the resulting supernatant (50 mg) were subjected to SDS-polyacrylamide gel electrophoresis (8% polyacrylamide) and the proteins subsequently transferred to polyvinylidene difluoride membranes by wet
transfer at 20 V over 24 h. The transfer buffer consisted of 48 mM Tris (pH 8.3), 39 mM glycine, 0.037% SDS, and 20% methanol. Membranes were subsequently incubated in blocking buffer (10 mM Tris, 100 mM NaCl, 3% bovine serum albumin and 0.1% Tween-20) followed by incubation with mouse-derived antisera against the key PTBR protein IBP (1:1000) (Sanofi Research, France) or b-actin (1:40,000) (Sigma, St. Louis, MO), corresponding to proteins of molecular weights of 18 and 42 kDa, respectively. Reblocking was followed by incubation with diluted horseradish peroxidase-coupled anti-mouse IgG (1:40,000) secondary antiserum. Each incubation step was of 1 h duration following which blots were washed several times with buffer (10 mM Tris, 100 mM NaCl, and 0.1% Tween-20). For the detection of specific antibody binding, the membranes were treated in accordance with the ECL-kit instructions and apposed to photosensitive X-OMAT film for 30 –60 s. Signal intensities were subsequently measured by densitometry using a microcomputer-based image display system (Imaging Research Inc., St. Catherines, Ontario). The linearity of the relationship between optical density and protein concentration was verified using appropriate standard curves. Blots were reversibly stained with Ponceau-S to determine uniformity of protein loading and evaluate protein transfer efficiency. For histological evaluation, coronal sections of brain of 40 mm thickness were cut at the levels of the lateral and medial globus pallidus (each group, n ¼ 5) using a vibratome and stained with cresyl violet for examination by light microscopy. Neuronal and glial cell numbers were assessed by counting two adjacent grid areas (0.36 mm2) at a magnification of 400 £ in each brain region and the values summed to yield counts for the globus pallidus. Blood samples were collected from rats used for RT-PCR following decapitation and the serum analyzed for total bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and creatinine using routine techniques. Samples of liver were also excised at the time of sacrifice (each group, n ¼ 4) and postfixed in 10% neutral buffered formalin. Paraffinembedded blocks of tissue were then prepared and sectioned. Mounted sections of liver were subsequently assessed for the presence of lesions by a trained pathologist (B.N.). Treatment with manganese resulted in an 81% increase in levels of the metal in the globus pallidus after 1 day of exposure (P , 0:05) which further increased to 551% after 4 days (P , 0:05) (Fig. 1). RT-PCR amplification of total mRNA yielded a product corresponding to ptbr which was expressed approximately 60% higher (P , 0:05) in rats treated with manganese for 4 days compared to controls (Fig. 2). Administration of manganese for 1 day, however, led to no change in ptbr expression. The increase in ptbr mRNA was calculated after normalization to b-actin mRNA levels. Consistent with the gene expression findings, manganese treatment for 1 day produced no change in the levels of IBP, an integral protein of the PTBR, but 4 days of
A.S. Hazell et al. / Neuroscience Letters 349 (2003) 13–16
Fig. 1. Levels of manganese in the globus pallidus following treatment with the metal. Exposure of rats to manganese for 1 (n ¼ 4) and 4 days (n ¼ 6) results in increased levels of the metal in the globus pallidus compared to control animals (n ¼ 6). Data are the means ^ SEM. *P , 0:05 compared with controls (one-way ANOVA with post-hoc Scheffe test).
exposure to this metal led to a 105% increase in its content in the globus pallidus by immunoblotting (Fig. 3). At the same time, the structure of the globus pallidus at the light microscope level appeared unremarkable with no evidence of neuronal cell loss (controls: 62 ^ 8; manganese (1 day): 56 ^ 6; manganese (4 days): 64 ^ 6) or gliosis. Manganese treatment for 4 days produced elevations of the enzymes
Fig. 2. Manganese treatment results in increased expression of PTBRs in the globus pallidus. Ptbr and b-actin were reverse-transcribed and amplified by RT-PCR. Results show representative bands: lane 1, molecular weight marker; lanes 3 and 4, control samples; lanes 5 and 6, manganese (1 day) samples; lanes 7 and 8, manganese (4 days); AMV reverse transcriptase was omitted in lanes 2 and 9 as negative controls for 1 and 4 day manganese groups, respectively. The graph shows quantitative analysis of ptbr mRNA expression with values normalized to b-actin levels. Data are means ^ SEM of control (n ¼ 6), manganese (1 day) (n ¼ 6), and manganese (4 days) (n ¼ 6) animals. *P , 0:05 compared with controls (one-way ANOVA with post-hoc Scheffe test).
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Fig. 3. Effect of manganese treatment on PTBR protein levels in the globus pallidus. Exposure to manganese results in an upregulation of PTBRs by immunoblotting. Results show representative immunoblots of IBP and bactin, in which IBP levels are increased after 4 days. Lanes were loaded with equal amounts of protein in each case (50 mg). Analysis shows densitometric results in globus pallidus of controls (n ¼ 4), manganese (1 day) (n ¼ 4), and manganese (4 days) (n ¼ 4) rats. Data are means ^ SEM. *P , 0:01 compared with controls (one-way ANOVA with post-hoc Scheffe test).
AST [controls: 122 ^ 10; manganese (1 day): 152 ^ 17; manganese (4 days): 325 ^ 32 IU/l, n ¼ 6; P , 0:05] and ALT [controls: 40 ^ 1; manganese (1 day): 65 ^ 15; manganese (4 days): 287 ^ 74, n ¼ 6; P , 0:05] in blood serum but levels of bilirubin and ALP activity were unchanged. Microscopic examination of the liver itself showed no obvious structural damage. In addition, manganese treatment had no effect on serum creatinine levels. The findings indicate that short-term (4 days) exposure to manganese resulted in increased ptbr expression accompanied by an upregulation of IBP in the globus pallidus. Although manganese accumulation in this brain region occurred as early as 1 day following commencement of treatment, this effect was not immediately reflected in an alteration of ptbr mRNA levels. Manganese is capable of binding to different forms of DNA structure [5], and may destabilize DNA duplexes leading to conformational changes within the nucleic acid structure and an alteration in the efficiency of gene transcription. However, such effects of manganese in the cell nucleus may require time to manifest themselves which may at least partly explain the absence of an early effect of this metal on PTBR mRNA and protein levels. Since manganese is normally excreted via the hepatobiliary route [12], it was necessary to assess the effects of manganese administration on liver function and histology. In addition, we also examined whether renal function might be affected in this model, as an alternative route for the removal of manganese is the kidneys. Our findings indicate that manganese treatment did not lead to serious liver toxicity. In addition, blood creatinine levels were
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unchanged, suggesting that renal integrity was unaffected following manganese treatment. In this study, we have focussed on the effect of short-term manganese treatment on the expression of PTBRs, a protein complex found particularly in astrocytes [2] that plays a major role in the production of neurosteroids in brain [10]. In an earlier study we demonstrated that exposure of cultured astrocytes to manganese resulted in an upregulation of binding sites for the PTBR ligand 3H-PK 11195 [3], consistent with the present findings. Increased PTBR binding sites may reflect alterations in the morphology, size, or number of mitochondria present in these astrocytes as a result of exposure to manganese. The consequences of such an upregulation of these receptors may be important in the pathophysiology of MN. Certain neurosteroids such as 3a-hydroxy-5a-pregnane20-one (allopregnanolone) bind with high affinity to the GABAA receptor for which they are positive modulators, i.e. they amplify the action of GABA and may also gate the chloride channel of the GABAA receptor in the absence of GABA [14]. Other neurosteroids such as pregnenolone sulfate exhibit negative modulatory effects on these receptors [6]. Thus, neurosteroids may alter neuronal excitability and therefore contribute to dysregulation of the functional integrity of the brain. Such an effect may also play a role in the extrapyramidal abnormality typical of MN. Other possible effects of PTBRs include an influence on mitochondrial morphology [15] and astrocytic oxidative metabolism [1]. In conclusion, upregulation of PTBRs is an early response to manganese exposure in the globus pallidus, a brain region that later develops profound neuronal loss in MN. Since Alzheimer type II astrocytosis is a pathological hallmark of chronic MN, and given the finding that treatment of mice with the neurosteroid tetrahydroprogesterone leads to the development of this cellular transformation [8], increased expression of PTBRs may be an important contributor to the pathophysiology of this brain disorder.
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
Acknowledgements These studies were funded by a grant from the Canadian Institutes of Health Research to A.S.H. (MOP-53110).
[15]
[16]
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modulators of intermediary metabolism, Trends Pharmacol. Sci. 7 (1986) 506 –511. R.R.H. Anholt, P.L. Pedersen, E.B. DeSouza, S.H. Snyder, The peripheral-type benzodiazepine receptor: localization to the mitochondrial outer membrane, J. Biol. Chem. 261 (1986) 576– 583. A.S. Hazell, P. Desjardins, R.F. Butterworth, Exposure of primary astrocyte cultures to manganese results in increased binding sites for the “peripheral-type” benzodiazepine receptor ligand 3H-PK 11195, Neurosci. Lett. 271 (1999) 5–8. A.S. Hazell, K.V. Rama Rao, D.V. Danbolt, D.V. Pow, R.F. Butterworth, Selective downregulation of the astrocyte glutamate transporters GLT-1 and GLAST within the medial thalamus in experimental Wernicke’s encephalopathy, J. Neurochem. 78 (2001) 560 –568. S.D. Kennedy, R.G. Bryant, Manganese-deoxyribonucleic acid binding modes. Nuclear magnetic relaxation dispersion results, Biophys. J. 50 (1986) 669 –676. M.D. Majewska, J.-M. Mienville, S. Vicini, Neurosteroid pregnenolone sulfate antagonizes electrophysiological responses to GABA in neurons, Neurosci. Lett. 90 (1988) 279–284. M.W. McEnery, A.M. Snowman, R.R. Trifiletti, S.H. Snyder, Isolation of the mitochondrial benzodiazepine receptor: association with the voltage-dependent anion channel and the adenine nucleotide carrier, Proc. Natl. Acad. Sci. USA 89 (1992) 3170–3174. M.D. Norenberg, Y. Itzhak, A.S. Bender, The peripheral benzodiazepine receptor and neurosteroids in hepatic encephalopathy, in: V. Felipo, S. Grisolı´a (Eds.), Advances in Cirrhosis, Hyperammonemia and Hepatic Encephalopathy, Plenum Press, New York, 1997, pp. 95– 111. U. Nudel, R. Zakut, M. Shani, S. Neuman, Z. Levy, D. Yaffe, The nucleotide sequence of the rat cytoplasmic beta-actin gene, Nucleic Acids Res. 11 (1983) 1759–1771. C.W. Olanow, P.F. Good, H. Shinotoh, K.A. Hewitt, F. Vingerhoets, B.J. Snow, M.F. Beal, D.B. Calne, D.P. Perl, Manganese intoxication in the rhesus monkey: a clinical, imaging, pathologic, and biochemical study, Neurology 46 (1996) 492 –508. V. Papadopoulos, Peripheral-type benzodiazepine/diazepam binding inhibitor receptor: biological role in steroidogenic cell function, Endocr. Rev. 14 (1993) 222 –240. P.S. Papavasiliou, S.T. Miller, G.C. Cotzias, Role of liver in regulating distribution and excretion of manganese, Am. J. Physiol. 211 (1966) 211 –216. A. Pentschew, F.F. Ebner, R.M. Kovatch, Experimental manganese encephalopathy in monkeys. A preliminary report, J. Neuropathol. Exp. Neurol. 22 (1963) 488 –499. G. Puia, M.R. Santi, S. Vicini, D.B. Pritchett, P.H. Seeburg, E. Costa, Differences in the negative allosteric modulation of GABA receptors elicited by 40 -chlorodiazepam and by a b-carboline-3-carboxylate ester: a study with natural and reconstituted receptors, Proc. Natl. Acad. Sci. USA 86 (1989) 7275–7279. T. Shiraishi, K.L. Black, K. Ikesaki, Peripheral benzodiazepine receptor ligands induce morphological changes in mitochondria of cultured glioma cells, Soc. Neurosci. Abstr. 16 (1990) 214. R. Sprengel, P. Werner, P.H. Seeburg, A.G. Mukhin, M.R. Santi, D.R. Grayson, A.A. Guidottti, K.E. Krueger, Molecular cloning and expression of cDNA encoding a peripheral-type benzodiazepine receptor, J. Biol. Chem. 264 (1989) 20415–20421.
Upregulation of ‘peripheral-type’ benzodiazepine receptors in the globus pallidus in a sub-acute rat model of manganese neurotoxicity Alan S. Hazella,*, Louise Normandina, Bich Nguyenb, Greg Kennedyc a b
Department of Medicine, Hoˆpital Saint-Luc, University of Montreal, Montreal, Quebec, Canada Department of Pathology, Hoˆpital Saint-Luc, University of Montreal, Montreal, Quebec, Canada c Department of Engineering Physics, E´cole Polytechnique, Montreal, Quebec, Canada Received 5 January 2003; accepted 13 May 2003
Abstract Manganese neurotoxicity (MN) is a neurological disorder characterized by selective neuronal loss in the globus pallidus together with characteristic morphological changes known as Alzheimer type II astrocytosis. In order to understand the underlying mechanisms responsible for these processes, we studied early effects of this metal in a sub-acute rat model. Levels of manganese in the globus pallidus were increased by 81% after 1 day of treatment and elevated by 551% compared to controls after 4 days. In addition, manganese treatment led to a 60% increase in ptbr expression, and a 105% increase in levels of its product, the isoquinoline-carboxamide binding protein, a major constituent of the ‘peripheral-type’ benzodiazepine receptor (PTBR) that is localized to astrocytes, in this brain region after 4 days. These results indicate that PTBRs, and possibly neurosteroids, are an early response to manganese exposure and may play a major role in the pathophysiology of MN. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: ‘Peripheral-type’ benzodiazepine receptor; Manganism; Astrocyte; Neurosteroid; Globus pallidus; Manganese neurotoxicity
Manganese is an essential element with an important role in the normal functioning of several enzymes. However, exposure to this metal can lead to manganese neurotoxicity (MN), in which neuronal loss in the globus pallidus [10] and the development of Alzheimer type II astrocytosis [13] are typical features. Alzheimer type II astrocytes have enlarged, pale nuclei with margination of chromatin and prominent nucleoli. The characteristic neuropathological finding of these abnormal cells suggests an important role for astrocytes in the pathophysiology of MN. Unlike central benzodiazepine receptors, the so-called ‘peripheral-type’ benzodiazepine receptor (PTBR) is expressed in high concentrations on the outer mitochondrial membrane, particularly of astrocytes [2]. Its basic structure consists of a 18 kDa isoquinoline carboxamide-binding protein (IBP), a 32 kDa voltage-sensitive anion channel, and a 30 kDa adenine nucleotide carrier [7]. Treatment of cultured astrocytes with manganese results in an upregulation of binding sites for the PTBR ligand 3H-PK 11195 [3]. * Corresponding author. Department of Medicine, Hoˆpital Saint-Luc (CHUM), 1058 St-Denis Street, Montreal, Quebec H2X 3J4, Canada. Tel.: þ 1-514-890-8310, ext. 35740; fax: þ1-514-412-7314. E-mail address: [email protected] (A.S. Hazell).
Although our understanding of the role of increased PTBRs in the pathogenesis of MN is unclear, PTBRs are an important source of neurosteroid production in the brain [11]. In this, the first of a series of investigations on the pathophysiology of MN, we have studied how manganese influences PTBR levels in a sub-acute rat model of this disorder. Male Sprague –Dawley rats (200 g) were placed in one of the following three groups: (A) manganese-treated group (1 day) (n ¼ 25); rats were administered a single dose of manganese (II) chloride (50 mg/kg body weight, i.p.) and allowed to survive for 24 h; (B) manganese-treated group (4 days) (n ¼ 27); rats were administered manganese (II) chloride (50 mg/kg body weight, i.p.) once per day for 4 days; (C) control group (n ¼ 27); rats were treated with carrier only (0.2 ml saline, i.p.). At the appropriate time, animals were decapitated and the brains removed with dissection of the lateral globus pallidus on dry ice, after which the tissue was stored at 2 80 8C until ready for study. Additional animals were perfusion-fixed as previously described [4] for subsequent neuropathological study. Manganese concentrations in brain samples were determined by instrumental neutron activation analysis. Brain
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00649-9
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A.S. Hazell et al. / Neuroscience Letters 349 (2003) 13–16
tissue samples were irradiated for a pre-determined time with the neutron flux of 5 £ 1011 neutron/cm2 per s in a Slowpoke nuclear reactor. After irradiation and before counting, each sample was transferred into a new plastic acid-rinsed vial so that Mn in the radioactive vial would not be detected. Energy emitted was detected as gamma-rays by a germanium detector and the average Mn concentration estimated. Total RNA was extracted from the globus pallidus using TRI Reagent (MRC Inc., Ohio) according to the manufacturer’s protocol. Putative contaminating DNA was eliminated by adding 100 units of RNase-free DNase I per 50 mg of total RNA at 37 8C for 1 h. Purified RNA was then extracted with phenol, precipitated with ethanol and resuspended in diethylpyrocarbonate-treated water. RNA samples were kept at 2 70 8C until use. Expression of the PTBR gene ptbr was investigated by reverse transcription-polymerase chain reaction (RT-PCR). b-Actin was used as an internal standard to monitor loading variations. Total RNA (1 mg) was mixed with 10 mM Tris – HCl (pH 8.3), 1.5 mM MgCl2, 50 mM KCl, 0.01% (w/v) bovine serum albumin, 100 mM dNTPs, primers at 1 mM each, AMV reverse transcriptase (80 units/ml), Taq DNA polymerase (20 units/ml) and 5 mCi/ml [a-32P]dCTP (3000 Ci/mM), for a total reaction volume of 50 ml. The reactions were initially heated at 50 8C for 15 min followed by PCR at 95 8C for 30 s, 59 8C for 1 min and 72 8C for 1 min. Ptbr and b-actin were amplified for 29 cycles and 15 cycles, respectively. Amplification efficiency conditions were determined after a kinetic study to ensure that the PCR product yields were linear with respect to the input RNA. After amplification, the samples were electrophoresed onto 8% polyacrylamide gels, dried, and autoradiographed at 2 70 8C with an intensifying screen. Each band was subsequently excised and Cerenkov radiation was quantitated using a bcounter. Oligonucleotide primers (Sheldon Biotechnology Center, McGill University, Quebec) were designed using the PRIME program (Genetic Computer Group, Wisconsin) based on the following GenBank accession numbers: ptbr, J05122 [16]; b-actin, V01217 [9]. The forward and reverse primer sequences were as follows: 50 -CCATGCTCAACTACTATGTATGGC-30 and 50 -GTACAACTGTCCCCGCATG-30 ( ptbr, 232 bp); 50 -CATCCCCCAAAGTTCTAC30 and 50 -CAAAGCCTTCATACATC-30 (b-actin, 347 bp). The monospecificity of oligonucleotide primers was verified using the nucleotide –nucleotide ‘blastn’ subprogram within BLAST (National Center for Biotechnology Information, Bethesda, MD). For immunoblotting, the lateral globus pallidus was extracted in buffer containing 50 mM Tris, 150 mM NaCl, 2% sodium dodecyl sulfate (SDS), 1% NP-40, 0.5% sodium deoxycholate (pH 8.0) and a protease inhibitor cocktail (Sigma, St. Louis, MO), and centrifuged at 10,000 £ g for 10 min at 4 8C. Aliquots of the resulting supernatant (50 mg) were subjected to SDS-polyacrylamide gel electrophoresis (8% polyacrylamide) and the proteins subsequently transferred to polyvinylidene difluoride membranes by wet
transfer at 20 V over 24 h. The transfer buffer consisted of 48 mM Tris (pH 8.3), 39 mM glycine, 0.037% SDS, and 20% methanol. Membranes were subsequently incubated in blocking buffer (10 mM Tris, 100 mM NaCl, 3% bovine serum albumin and 0.1% Tween-20) followed by incubation with mouse-derived antisera against the key PTBR protein IBP (1:1000) (Sanofi Research, France) or b-actin (1:40,000) (Sigma, St. Louis, MO), corresponding to proteins of molecular weights of 18 and 42 kDa, respectively. Reblocking was followed by incubation with diluted horseradish peroxidase-coupled anti-mouse IgG (1:40,000) secondary antiserum. Each incubation step was of 1 h duration following which blots were washed several times with buffer (10 mM Tris, 100 mM NaCl, and 0.1% Tween-20). For the detection of specific antibody binding, the membranes were treated in accordance with the ECL-kit instructions and apposed to photosensitive X-OMAT film for 30 –60 s. Signal intensities were subsequently measured by densitometry using a microcomputer-based image display system (Imaging Research Inc., St. Catherines, Ontario). The linearity of the relationship between optical density and protein concentration was verified using appropriate standard curves. Blots were reversibly stained with Ponceau-S to determine uniformity of protein loading and evaluate protein transfer efficiency. For histological evaluation, coronal sections of brain of 40 mm thickness were cut at the levels of the lateral and medial globus pallidus (each group, n ¼ 5) using a vibratome and stained with cresyl violet for examination by light microscopy. Neuronal and glial cell numbers were assessed by counting two adjacent grid areas (0.36 mm2) at a magnification of 400 £ in each brain region and the values summed to yield counts for the globus pallidus. Blood samples were collected from rats used for RT-PCR following decapitation and the serum analyzed for total bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and creatinine using routine techniques. Samples of liver were also excised at the time of sacrifice (each group, n ¼ 4) and postfixed in 10% neutral buffered formalin. Paraffinembedded blocks of tissue were then prepared and sectioned. Mounted sections of liver were subsequently assessed for the presence of lesions by a trained pathologist (B.N.). Treatment with manganese resulted in an 81% increase in levels of the metal in the globus pallidus after 1 day of exposure (P , 0:05) which further increased to 551% after 4 days (P , 0:05) (Fig. 1). RT-PCR amplification of total mRNA yielded a product corresponding to ptbr which was expressed approximately 60% higher (P , 0:05) in rats treated with manganese for 4 days compared to controls (Fig. 2). Administration of manganese for 1 day, however, led to no change in ptbr expression. The increase in ptbr mRNA was calculated after normalization to b-actin mRNA levels. Consistent with the gene expression findings, manganese treatment for 1 day produced no change in the levels of IBP, an integral protein of the PTBR, but 4 days of
A.S. Hazell et al. / Neuroscience Letters 349 (2003) 13–16
Fig. 1. Levels of manganese in the globus pallidus following treatment with the metal. Exposure of rats to manganese for 1 (n ¼ 4) and 4 days (n ¼ 6) results in increased levels of the metal in the globus pallidus compared to control animals (n ¼ 6). Data are the means ^ SEM. *P , 0:05 compared with controls (one-way ANOVA with post-hoc Scheffe test).
exposure to this metal led to a 105% increase in its content in the globus pallidus by immunoblotting (Fig. 3). At the same time, the structure of the globus pallidus at the light microscope level appeared unremarkable with no evidence of neuronal cell loss (controls: 62 ^ 8; manganese (1 day): 56 ^ 6; manganese (4 days): 64 ^ 6) or gliosis. Manganese treatment for 4 days produced elevations of the enzymes
Fig. 2. Manganese treatment results in increased expression of PTBRs in the globus pallidus. Ptbr and b-actin were reverse-transcribed and amplified by RT-PCR. Results show representative bands: lane 1, molecular weight marker; lanes 3 and 4, control samples; lanes 5 and 6, manganese (1 day) samples; lanes 7 and 8, manganese (4 days); AMV reverse transcriptase was omitted in lanes 2 and 9 as negative controls for 1 and 4 day manganese groups, respectively. The graph shows quantitative analysis of ptbr mRNA expression with values normalized to b-actin levels. Data are means ^ SEM of control (n ¼ 6), manganese (1 day) (n ¼ 6), and manganese (4 days) (n ¼ 6) animals. *P , 0:05 compared with controls (one-way ANOVA with post-hoc Scheffe test).
15
Fig. 3. Effect of manganese treatment on PTBR protein levels in the globus pallidus. Exposure to manganese results in an upregulation of PTBRs by immunoblotting. Results show representative immunoblots of IBP and bactin, in which IBP levels are increased after 4 days. Lanes were loaded with equal amounts of protein in each case (50 mg). Analysis shows densitometric results in globus pallidus of controls (n ¼ 4), manganese (1 day) (n ¼ 4), and manganese (4 days) (n ¼ 4) rats. Data are means ^ SEM. *P , 0:01 compared with controls (one-way ANOVA with post-hoc Scheffe test).
AST [controls: 122 ^ 10; manganese (1 day): 152 ^ 17; manganese (4 days): 325 ^ 32 IU/l, n ¼ 6; P , 0:05] and ALT [controls: 40 ^ 1; manganese (1 day): 65 ^ 15; manganese (4 days): 287 ^ 74, n ¼ 6; P , 0:05] in blood serum but levels of bilirubin and ALP activity were unchanged. Microscopic examination of the liver itself showed no obvious structural damage. In addition, manganese treatment had no effect on serum creatinine levels. The findings indicate that short-term (4 days) exposure to manganese resulted in increased ptbr expression accompanied by an upregulation of IBP in the globus pallidus. Although manganese accumulation in this brain region occurred as early as 1 day following commencement of treatment, this effect was not immediately reflected in an alteration of ptbr mRNA levels. Manganese is capable of binding to different forms of DNA structure [5], and may destabilize DNA duplexes leading to conformational changes within the nucleic acid structure and an alteration in the efficiency of gene transcription. However, such effects of manganese in the cell nucleus may require time to manifest themselves which may at least partly explain the absence of an early effect of this metal on PTBR mRNA and protein levels. Since manganese is normally excreted via the hepatobiliary route [12], it was necessary to assess the effects of manganese administration on liver function and histology. In addition, we also examined whether renal function might be affected in this model, as an alternative route for the removal of manganese is the kidneys. Our findings indicate that manganese treatment did not lead to serious liver toxicity. In addition, blood creatinine levels were
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unchanged, suggesting that renal integrity was unaffected following manganese treatment. In this study, we have focussed on the effect of short-term manganese treatment on the expression of PTBRs, a protein complex found particularly in astrocytes [2] that plays a major role in the production of neurosteroids in brain [10]. In an earlier study we demonstrated that exposure of cultured astrocytes to manganese resulted in an upregulation of binding sites for the PTBR ligand 3H-PK 11195 [3], consistent with the present findings. Increased PTBR binding sites may reflect alterations in the morphology, size, or number of mitochondria present in these astrocytes as a result of exposure to manganese. The consequences of such an upregulation of these receptors may be important in the pathophysiology of MN. Certain neurosteroids such as 3a-hydroxy-5a-pregnane20-one (allopregnanolone) bind with high affinity to the GABAA receptor for which they are positive modulators, i.e. they amplify the action of GABA and may also gate the chloride channel of the GABAA receptor in the absence of GABA [14]. Other neurosteroids such as pregnenolone sulfate exhibit negative modulatory effects on these receptors [6]. Thus, neurosteroids may alter neuronal excitability and therefore contribute to dysregulation of the functional integrity of the brain. Such an effect may also play a role in the extrapyramidal abnormality typical of MN. Other possible effects of PTBRs include an influence on mitochondrial morphology [15] and astrocytic oxidative metabolism [1]. In conclusion, upregulation of PTBRs is an early response to manganese exposure in the globus pallidus, a brain region that later develops profound neuronal loss in MN. Since Alzheimer type II astrocytosis is a pathological hallmark of chronic MN, and given the finding that treatment of mice with the neurosteroid tetrahydroprogesterone leads to the development of this cellular transformation [8], increased expression of PTBRs may be an important contributor to the pathophysiology of this brain disorder.
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
Acknowledgements These studies were funded by a grant from the Canadian Institutes of Health Research to A.S.H. (MOP-53110).
[15]
[16]
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