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Ischemia induces metallothionein III expression in neurons of rat brain
Life Sciences, Vol. 64, No. 8, pp. 707-715, 1999 Copyright0 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0024-3205/99 $19.00 + .OO
PI1 SOO24-3205(98)00612-Z
ELSEVIER
ISCHEMIA
INDUCES METALLOTHIONEIN III EXPRESSION IN NEURONS OF RAT BRAIN
Shingo Yanagitani’, Hiroaki Miyazaki’, Yoshitsugu Nakahashi’*, Kenji Kuno’, Yohji Ueno’, Masanori Matsushita’, Yuji Naitoh’, Shigeru Taketan? and Kyoichi Inoue’
‘Third Department of Internal Medicine and ‘Department of Hygiene, Kansai Medical University, Fumizonocho, Moriguchi, Osaka 570-8506, Japan (Received in final form November 13, 1998)
Summary Metallothionein III (MT-III) is a brain-specific member of the metallothionein family and binds zinc in vivo. In order to confirm the precise localization of MTIII in normal rat brain and the change of MT-III expression after transient whole brain ischemia, we raised a high affinity phagemid-antibody specific for rat MT-III. Immunohistochemical analysis revealed that MT-III in normal brain is localized abundantly in neuronal cell bodies in CAl-3 regions of hippocampus, dentate gyms, cerebral cortex, olfactory bulb and Purkinje cells in cerebellum. This expression pattern of MT-III was similar to that of MT-III mRNA observed by in situ hybridization studies. ELISA and Northern blot analysis revealed that MT-III protein as well as mRNA levels were up-regulated in cerebrum soon after ischemic stress. Immunohistochemical analysis also demonstrated intense staining in neurons in injured brain after ischemia, which distributed in the same regions as in normal brain. These results suggest that MT-III plays an important role in protecting neurons from ischemic insult by reducing neurotoxic zinc levels and inhibits uncontrolled growth of neurites after ischemia. Key Words: metallothionein immunohistochemistry
III, ischemia, phagemid-antibody,
zinc, Western blot, ELISA, Northern blot,
Metallothioneins (MTs) are a family of low-molecular-weight (6800 Da), cysteine rich intracellular metal-binding proteins that are present ubiquitously in a wide variety of species (1,2). In mammals, two isoforms, MT-I and MT-II, are expressed coordinately in most organs * Corresponding author: Yoshitsugu Nakahashi, Third Department of Internal Medicine, Kansai Medical University, Fumizonocho, Moriguchi, Osaka 570-8506, Japan. Phone: +81-6-9921001. Fax number: +81-6-997-5490.
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and they are proposed to play an important role in the detoxification of heavy metals, homeostasis of essential metals and protection against oxygen radical damage (l-3). MT-III is a brain-specific member of the MT family (3-5) and binds zinc in vivo (6) and in vitro (7). Human, mouse and rat MT-III are very similar to MT-I and MT-II except for the insertion of a threonine residue after the fifth amino acid and the insertion of an amino acid block near the carboxyl terminus (4,5,8,9). MT-I and MT-II expression is regulated by a wide variety of metals, hormones and xenobiotics, while MT-III responds poorly, or not at all, to these stimuli (2,3,5). In addition, MT-III, initially termed growth inhibitory factor, has the ability to inhibit the survival of rat cultured neurons (4). This inhibitory activity was not mimicked by either MT-I or MT-II (3,4). In situ hybridization studies revealed that MT-III mRNA is expressed exclusively in neurons of hippocampus, cortex and amygdala (6,10,11) and the localization of MT-III is similar to that of neurons that contain highly concentrated zinc (3,6). In contrast to these studies, MT-III was detected most abundantly in the astrocytes by immunohistochemical analysis (4,12). Thus, the distribution of the MT-III-producing‘cells remains controversial. Therefore, we raised a high affinity phagemid-antibody for MT-III instead of conventional Here we report the distribution of MT-III in normal rat brain by antibodies. Furthermore, the change of MT-III expression after rat immunohistochemical analysis. transient whole brain ischemia was examined using ELISA and RNA blots in order to evaluate The distribution of MT-III by immunohistochemical analysis the influence of ischemic stress. in injured rat brain after ischemia was also shown.
Methods Experimental model : A four-vessel occlusion procedure was performed to obtain transient whole brain ischemia by the method of Pulsinelli and Brierly (13). Eight male Wistar rats weighing between 270-3008 were decapitated on days 1, 2, 3, 4, 7 and 14 after brain ischemia. Control animals were pretreated in the same way and decapitated the next day without clipping of the carotid arteries. pSKAN PhagemidPreparation of monoclonal phagemid-antibody specific for rat MT-III : antibodies specific for rat MT-III were raised in accordance with the manufacture’s directions (MoBiTec, Gottingen, Germany) (14). After selection of high affinity phagemid-antibodies, hypervariable regions of each phagemid were subcloned into TA cloning vector (Invitrogen, Carlsbad, CA) and sequenced (15). To obtain high affinity monoclonal phagemid-antibody, the hypervariable region containing a major DNA sequence was ligated into all of three hypervariable regions in one phagemid. MT-I, -11 : Preparation of recombinant MT-I, -II and -IIIproteins and Western blot analysis Each PCR product was and -111 cDNAs of the entire coding regions were cloned by RT-PCR. ligated into pGEX 4T expression vector respectively, and GST fusion proteins of MT-I, -II and III were expressed in E. coli and purified according to the manufacture’s instructions (Pharmacia Then, GST-MTs fusion proteins were cleaved by thrombin. Biotech, Uppsala, Sweden). Western blot analysis was performed as described previously (15). Briefly, MT-I, -II, and -III a 15% SDS-polyacrylamide gel and recombinant proteins (10 ti g) were separated on transferred onto a Hybond ECL membrane (Amersham, Arlington Heights, IL). To detect the
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immunoreactive bands of MT-III, the membrane was incubated with the obtained monoclonal phagemid-antibody specific for rat MT-III as a primary antibody and sheep anti-Ml3 phage polyclonal affinity purified antibody horseradish peroxidase conjugated (Pharmacia Biotech) as a We used the anti-metallothionein antibody E9 (DAKO, Carpinteria, CA) as a second antibody. primary antibody and goat anti mouse IgG polyclonal antibody horseradish peroxidase conjugated (DAKO) as a second antibody to detect the immunoreactive bands of MT-I or MT-II. The immunoreactive bands were visualized using ECL chemiluminescent substrate (Amersham). Immunohistochemical analysis : Coronal sections of frozen tissue from normal and ischemic injured rat brains were cut at 4 fi m thickness and immunostained as described previously (16). The obtained monoclonal phagemid-antibody was used as primary antibody and sheep anti-Ml3 polyclonal affinity purified antibody horseradish peroxidase conjugated was applied as second antibody. Immunostaining was developed using HistoMark Black (Kirkegaard and Perry Laboratories, Gaitherrsburg, MD). Northern blot analysis : Total RNA was isolated from tissue samples as described previously Northern blot analysis was performed by using digoxigenin-labeled probes according to (15). the manufacture’s instructions (Boehringer Mannheim GmbH, Mannheim, Germany). A specific probe for MT-III (nucleotide position #173 to #284) was obtained by cleaving rat MTIII cDNA with Mbo II and Nci I. The density of reacted band was measured at 685nm with FluorImager (Molecular Dynamics, Sunnyvale, CA). Levels of mRNA were expressed as a ratio relative to sham-operated rats. Enzyme-linked immunosorbent assay (ELLSA) using phagemid-antibody : MT-III expression ELISA was carried out by the method of Tesar et al (17). In brief, was measured by ELISA. 96-well microtiter plates (Becton Dickison, NJ) were coated with brain homogenates prepared as was used as primary antibody and sheep described (4). The monoclonal phagemid-antibody anti-Ml3 polyclonal affinity purified antibody horseradish peroxidase conjugated was applied as They were stained with o-phenylenediamine sulfate solution and 0.03% H,O, second antibody. in O.lM citric acid and 0.2M Na,HPO,. The color development was measured at 492 nm with an ELISA-reader model Spectra max 250 (Molecular Devices, Sunnyvale, CA). Levels of MTIII were expressed as a ratio relative to sham-operated rats.
Results DNA sequencing of the We raised high affinity phagemid-antibodies specific for rat MT-III. hypervariable region in the high affinity phagemid with rat MT-III was carried out. The major sequence was 5’-cagagggggagggtttagacgcgg-3’, which translated into eight amino acids; Q-R-GWe subcloned this DNA fragment into all of the hypervariable regions in one R-V-S-T-R. phagemid to obtain the monoclonal phagemid-antibody. As shown in Fig. 1, the obtained monoclonal phagemid-antibody reacted with only rat MT-III at the expected position (7.2 kD) By contrast, anti-metallothionein antibody E9 reacted with rat MT-I but not rat MT-I or MT-II. and MT-II, but not MT-III. The time course of MT-III expression after rat transient whole brain ischemia was investigated Four days after ischemic stress, the expression of MT-III in the cerebrum increased by ELISA.
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Induction of MT-III after Ischemia
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to about 2.2-fold that of control @
A
6
kDs B E 4.-32 -
lb
-
7.2 -
1
2
3
1
2
3
Fig. 1 Western blot analysis of MT-I, -11 and -111. 10 ,Y g of MT-I, -11, and -III recombinant proteins were electrophoresed on 15% SDS-polyacrylamide gel, transferred to nitrocellulose membrane and then immunostained with the monoclonal phagemid-antibody (A) or anti-metallothionein antibody E9 (B). Lane 1, MT-I; lane 2, MT-II; lane 3, MT-III.
Induction of MT-III after Ischemia
Vol. 64, No. 8, 1999
A 2.5
*
2
**
1.5 1 0.5iJ@ays)
shemc;
sham
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,,,,,
2
3
4
7
14 (days)
C 2.5 *
2
*
1.5 1 0.5ifi sham
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14 (days)
Fig. 2 Time course of MT-III protein and mRNA after transient whole brain ischemia. Cerebral protein level (A) and cerebellum protein level (B) was measured by ELISA with the monoclonal phagemid-antibody specific for rat MT-III. Whole brain mRNA level (C) was measured by Northern blot analysis. Levels of protein or mRNA were expressed as a ratio relative to sham-operated rats. Each point represents the mean + SD. P-value against sham control analyzed with Student’s t test. *p
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Fig. 3 Immunohistochemical analysis for MT-III in rat brain. Coronal sections from normal and ischemic injured rat brains were immunostained with the monoclonal phagemid-antibody. Normal cerebrum at low magnification ( X 4) (A) and hippocampus at high magnification (X80) (C), and normal cerebellum at low magnification (X 32) (E) and at high magnification (X 50) (F). (B) and (D) show the same cerebral regions as in (A) and (C) after transient whole brain ischemia. Hi, hippocampus; DG, dentate gyrus; Cx, cortex; T, thalamus; CP, caudate putamen; Am, amygdala; Pl, pyramidal cell layer; Ml, molecular layer; Pu, Purkinje cell layer; Gl, granular layer. Scale bars, lmm (A and B); 50 pm (C, D, E and F).
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Discussion The present study demonstrated that MT-III in normal rat brain is exclusively localized in neurons in hippocampus, dentate gyrus, cerebral cortex, olfactory bulb and Purkinje cells in cerebellum. These results are consistent with previous observations by in situ hybridization studies (6,10,11) that MT-III mRNA is expressed in neuronal cells. The present data also support the observations using mouse MT-III transgenic mice that the MT-III transgene was present in readily identifiable neurons, but not glia (6). In contrast, Tsuji et al. (4,12) reported that the MT-III immunoreactivity was localized most abundantly in the astrocytes of normal human, rat and mouse brain using their own antibodies. The reason for this discrepancy is not clear, but it is possible that their antibodies were not specific for rat MT-III since a rabbit polyclonal antibody against a synthetic polypeptide homologous to the unique four amino acid block near the carboxyl terminus of rat MT-III (4,18) and a monoclonal antibody against human (12), not rat MT-III, were used. Antibodies have problems targeting small epitopes due to steric hindrance by their surface peptides chains. On the other hand, phagemid-antibodies are able to target very small epitopes, because they search for binding sites with only a fingertip rather than two whole hands (17). Rat MT-III has high similarity to rat MT-I and MT-II (66% and 62% identity in amino acid sequence, respectively) (9). Therefore, in this study, we raised a high affinity phagemidantibody for rat MT-III. The phagemid-antibody reacted with only rat MT-III, but not rat MT-I or MT-II, by Western blot analysis (Fig. 1). Immunohistochemical analysis with the phagemidantibody showed that MT-III immunoreactivity is present abundantly in neuronal cell bodies in CAl-3 regions of hippocampus, the dentate gyrus, cerebral cortex, olfactory bulb and Purkinje cells in cerebellum (Fig. 3A,C,E,F). This finding agrees with our observations by in situ hybridization study (unpublished data). The distribution of MT-III shown in this study (Fig. 3A,C,E,F) was similar to that of histochemically reactive zinc as detected with Timm’s stain or 6-methoxy-8-para-toluene sulfonamide quinoline (TSQ) histofluorescence (6). Several studies showed that zinc is neurotoxic (19,20) and its intracellular accumulation may trigger neuronal death caused by seizures (21) and transient cerebral ischemia (22). In addition, MT-III binds zinc in vivo (6) and confers resistance to zinc cytotoxity in cultured cells transfected with MT-III cDNA (23). Erickson et al. (24) reported that MT-III-deficient mice were sensitive to seizure-induced injury to CA3 hippocampal neurons, whereas transgenic mice expressing elevated levels of MT-III were resistant to this damage. Thus, MT-III in hippocampal neurons may have a potential role in detoxification of zinc during neuronal stress. Recently, Yuguchi et al. (10) reported that MT-III mRNA expression increased transiently in neurons of the rat cerebral cortex on day 4 after focal cerebral ischemia. In the present study, ELISA and Northern blot analysis revealed that the expression of MT-III protein as well as mRNA increased in cerebrum on day 4 after ischemic stress (Fig. 2A,C). And immunohistochemical analysis demonstrated intense staining in neurons in injured brain after ischemia, which distributed in the same regions as in normal brain (Fig. 3B,D). After transient cerebral ischemia in rats, zinc accumulated specifically in degenerating neurons in the hippocampus as well as in the cerebral cortex, thalamus, striatum and amygdala (22). We demonstrated that MT-III is up-regulated and expressed at high levels in neurons of cerebrum
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soon after ischemic insult (Fig. 3D). The pattern of the elevated expression of MT-III was similar to that of accumulated zinc. These findings indicate that MT-III is induced only in neurons by ischemic stress and suggest that MT-III is involved in protecting neurons from ischemic insult by reducing the level of neurotoxic zinc. Another specific function of MT-III is growth inhibition of neurons. MT-III inhibits the survival and neurite formation of cortical neurons in vitro (4). High level expression of MT-III in neurons after ischemic insult may prevent neuronal sprouting and inhibit uncontrolled overgrowth of neurites. MT-III levels were reduced in the cerebral cortex of Alzheimer’s disease (AD) brains (4,s). Uchida (25) postulated that a deficit of MT-III in AD brains could lead to excessive stimulation of neurons, resulting in neuronal cell death and that MT-III might be associated with some neurodegenerative disorders. In contrast, Erickson et aZ. (26) reported that neither MT-III mRNA nor protein were significantly decreased in AD brains and that MT-III down-regulation is not an important pathologic event in AD. Therefore, the implication of MT-III in AD pathology is still controversial. The present study demonstrates that MT-III in neurons plays an important role in the central nervous system (CNS) in normal and injured brains. We hypothesize that neurons themselves prevent abortive neuronal sprouting by expressing MT-III to maintain the homeostasis of zinc in the CNS. The specific phagemid-antibody for MT-III raised in this study will be useful to clarify the neurophysiological functions of MT-III in the CNS and neuropathogenic potential in AD.
Acknowledgements We thank Ms. K. Yasaka for excellent technical assistance. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan.
References 1. 2. 3. 4. 5. 6.
7.
J.H.R. dG1 and A. SCdFFER, Biochemistry 27 8509-8515 (1988). R.D. PALMITER, Experientia Suppl. 52 63-80 (1987). M. ASCHNER, M.G. CHERIAN, C.D. KLAASSEN, R.D. PALMITER, J.C. ERICKSON and A.I. BUSH, Toxicol. Appl. Pharmacol. 142 229-242 (1997). Y. UCHIDA, K. TAKIO, K. TITANI, Y. IHARA and M. TOMONAGA, Neuron 7 337-347 (1991). R.D. PALMITER, S.D. FINDLEY, T.E. WHITMORE and D.M. DURMAM, Proc. Natl. Acad. Sci. USA 89 6333-6337 (1992). B.A. MASTERS, C.J. QUAIFE, J.C. ERICKSON, E.J. KELLY, G.J. FROELICK, B.P. ZAMBROWICZ, R.L. BRINSTER and R.D. PALMITER, J. Neurosci. 14 5844-5857 (1994). A.K. SEWELL, L.T. JENSEN, J.C. ERICKSON, R.D. PALMITER and D.R. WINGE, Biochemistry 34 4740-4747 (1995).
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8. 9. 10.
11.
12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.
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S. TSUJI, H. KOBAYASHI, Y. UCHIDA, Y. IHARA and T. Miyatake, EMBO J. 1148434850 (1992). H. KOBAYASHI, Y. UCHIDA, Y. IHARA, K. NAKAJIMA, S. KOHSAKA, T. MIYATAKE and S. TSUJI, Mol. Brain Res. 19 188-194 (1993). T. YUGUCHI, E. KOHMURA, T. SAKAKI, M. NONAKA, K. YAMADA, T. YAMASHITA, T. KISHIGUCHI, T. SAKAGUCHI and T. HAYAKAWA, J. Cereb. Blood Flow Metab. 17 745-752 (1997). S. CHOUDHURI, K.K. KRAMER, N.E.J. BERMAN, T.P. DALTON, G.K. ANDREWS and C.D. KLAASSEN, Toxicol. Appl. Pharmacol. 131 144-154 (1995). I. HOZUMI, T. INUZUKA, H. ISHIGURO, M. HIRAIWA, Y. UCHIDA and S. TSUJI, Brain Res. 741 197-204 (1996). W.A. PULSINELLI and J.B. BRIERLEY, Stroke 10 267-272 (1979). P. R&ITGEN and J. COLLINS, Gene 164 243-250 (1995). Y. NAKAHASHI, H. FUJITA, S.TAKETANI, N. ISHIDA, A. KAPPAS and S. SASSA, Proc. Natl. Acad. Sci. USA 89 281-285 (1992). B. JASANI and M. E. ELMES, Methods in Enzymology 205 95-107 (1991). M. TESAR, C. BECKMANN, P. RGTTGEN, B. HAASE, U. FAUDE and K.N. TIMMIS, Immunotechnology 153-64 (1995). I. HOZUMI, T. INUZUKA, M. HIRAIWA, Y. UCHIDA, T. ANEZAKI, H. ISHIGURO, H. KOBAYASHI, Y. UDA, T. MIYATAKE and S. TSUJI, Brain Res. 688 143-148 (1995). M. YOKOYAMA, J. KOH and D.W. CHOI, Neurosci. Lett. 71351-355 (1986). D.W. CHOI, M. YOKOYAMA and J. KOH, Neuroscience 24 67-79 (1988). C.J. FREDERICKSON, M.D. HERNANDEZ and J.F. McGINTY, Brain Res. 480 317-321 (1989). J.Y. KOH, S.W. SUH, B.J. GWAG, Y.Y. HE, C.Y. HSU and D.W. CHOI, Science 272 1013-1016 (1996). R.D. PALMITER, Toxicol. Appl. Pharmacol. 135 139-146 (1995). J.C. ERICKSON, G. HOLLOPETER, S.A. THOMAS, G.J. FROELICK and R.D. PALMITER, J. Neurosci. 17 1271-1281(1997). Y. UCHIDA, Biol. Signals 3 211-215 (1994). J.C. ERICKSON, A.K. SEWELL, L.T. JENSEN, D.R. WINGE and R.D. PALMITER, Brain Res. 649 297-304 (1994).
PI1 SOO24-3205(98)00612-Z
ELSEVIER
ISCHEMIA
INDUCES METALLOTHIONEIN III EXPRESSION IN NEURONS OF RAT BRAIN
Shingo Yanagitani’, Hiroaki Miyazaki’, Yoshitsugu Nakahashi’*, Kenji Kuno’, Yohji Ueno’, Masanori Matsushita’, Yuji Naitoh’, Shigeru Taketan? and Kyoichi Inoue’
‘Third Department of Internal Medicine and ‘Department of Hygiene, Kansai Medical University, Fumizonocho, Moriguchi, Osaka 570-8506, Japan (Received in final form November 13, 1998)
Summary Metallothionein III (MT-III) is a brain-specific member of the metallothionein family and binds zinc in vivo. In order to confirm the precise localization of MTIII in normal rat brain and the change of MT-III expression after transient whole brain ischemia, we raised a high affinity phagemid-antibody specific for rat MT-III. Immunohistochemical analysis revealed that MT-III in normal brain is localized abundantly in neuronal cell bodies in CAl-3 regions of hippocampus, dentate gyms, cerebral cortex, olfactory bulb and Purkinje cells in cerebellum. This expression pattern of MT-III was similar to that of MT-III mRNA observed by in situ hybridization studies. ELISA and Northern blot analysis revealed that MT-III protein as well as mRNA levels were up-regulated in cerebrum soon after ischemic stress. Immunohistochemical analysis also demonstrated intense staining in neurons in injured brain after ischemia, which distributed in the same regions as in normal brain. These results suggest that MT-III plays an important role in protecting neurons from ischemic insult by reducing neurotoxic zinc levels and inhibits uncontrolled growth of neurites after ischemia. Key Words: metallothionein immunohistochemistry
III, ischemia, phagemid-antibody,
zinc, Western blot, ELISA, Northern blot,
Metallothioneins (MTs) are a family of low-molecular-weight (6800 Da), cysteine rich intracellular metal-binding proteins that are present ubiquitously in a wide variety of species (1,2). In mammals, two isoforms, MT-I and MT-II, are expressed coordinately in most organs * Corresponding author: Yoshitsugu Nakahashi, Third Department of Internal Medicine, Kansai Medical University, Fumizonocho, Moriguchi, Osaka 570-8506, Japan. Phone: +81-6-9921001. Fax number: +81-6-997-5490.
708
Induction of MT-III after Ischemia
Vol. 64, No. 8, 1999
and they are proposed to play an important role in the detoxification of heavy metals, homeostasis of essential metals and protection against oxygen radical damage (l-3). MT-III is a brain-specific member of the MT family (3-5) and binds zinc in vivo (6) and in vitro (7). Human, mouse and rat MT-III are very similar to MT-I and MT-II except for the insertion of a threonine residue after the fifth amino acid and the insertion of an amino acid block near the carboxyl terminus (4,5,8,9). MT-I and MT-II expression is regulated by a wide variety of metals, hormones and xenobiotics, while MT-III responds poorly, or not at all, to these stimuli (2,3,5). In addition, MT-III, initially termed growth inhibitory factor, has the ability to inhibit the survival of rat cultured neurons (4). This inhibitory activity was not mimicked by either MT-I or MT-II (3,4). In situ hybridization studies revealed that MT-III mRNA is expressed exclusively in neurons of hippocampus, cortex and amygdala (6,10,11) and the localization of MT-III is similar to that of neurons that contain highly concentrated zinc (3,6). In contrast to these studies, MT-III was detected most abundantly in the astrocytes by immunohistochemical analysis (4,12). Thus, the distribution of the MT-III-producing‘cells remains controversial. Therefore, we raised a high affinity phagemid-antibody for MT-III instead of conventional Here we report the distribution of MT-III in normal rat brain by antibodies. Furthermore, the change of MT-III expression after rat immunohistochemical analysis. transient whole brain ischemia was examined using ELISA and RNA blots in order to evaluate The distribution of MT-III by immunohistochemical analysis the influence of ischemic stress. in injured rat brain after ischemia was also shown.
Methods Experimental model : A four-vessel occlusion procedure was performed to obtain transient whole brain ischemia by the method of Pulsinelli and Brierly (13). Eight male Wistar rats weighing between 270-3008 were decapitated on days 1, 2, 3, 4, 7 and 14 after brain ischemia. Control animals were pretreated in the same way and decapitated the next day without clipping of the carotid arteries. pSKAN PhagemidPreparation of monoclonal phagemid-antibody specific for rat MT-III : antibodies specific for rat MT-III were raised in accordance with the manufacture’s directions (MoBiTec, Gottingen, Germany) (14). After selection of high affinity phagemid-antibodies, hypervariable regions of each phagemid were subcloned into TA cloning vector (Invitrogen, Carlsbad, CA) and sequenced (15). To obtain high affinity monoclonal phagemid-antibody, the hypervariable region containing a major DNA sequence was ligated into all of three hypervariable regions in one phagemid. MT-I, -11 : Preparation of recombinant MT-I, -II and -IIIproteins and Western blot analysis Each PCR product was and -111 cDNAs of the entire coding regions were cloned by RT-PCR. ligated into pGEX 4T expression vector respectively, and GST fusion proteins of MT-I, -II and III were expressed in E. coli and purified according to the manufacture’s instructions (Pharmacia Then, GST-MTs fusion proteins were cleaved by thrombin. Biotech, Uppsala, Sweden). Western blot analysis was performed as described previously (15). Briefly, MT-I, -II, and -III a 15% SDS-polyacrylamide gel and recombinant proteins (10 ti g) were separated on transferred onto a Hybond ECL membrane (Amersham, Arlington Heights, IL). To detect the
Vol. 64, No. 8, 1999
Induction of MT-III after Ischemia
IQ9
immunoreactive bands of MT-III, the membrane was incubated with the obtained monoclonal phagemid-antibody specific for rat MT-III as a primary antibody and sheep anti-Ml3 phage polyclonal affinity purified antibody horseradish peroxidase conjugated (Pharmacia Biotech) as a We used the anti-metallothionein antibody E9 (DAKO, Carpinteria, CA) as a second antibody. primary antibody and goat anti mouse IgG polyclonal antibody horseradish peroxidase conjugated (DAKO) as a second antibody to detect the immunoreactive bands of MT-I or MT-II. The immunoreactive bands were visualized using ECL chemiluminescent substrate (Amersham). Immunohistochemical analysis : Coronal sections of frozen tissue from normal and ischemic injured rat brains were cut at 4 fi m thickness and immunostained as described previously (16). The obtained monoclonal phagemid-antibody was used as primary antibody and sheep anti-Ml3 polyclonal affinity purified antibody horseradish peroxidase conjugated was applied as second antibody. Immunostaining was developed using HistoMark Black (Kirkegaard and Perry Laboratories, Gaitherrsburg, MD). Northern blot analysis : Total RNA was isolated from tissue samples as described previously Northern blot analysis was performed by using digoxigenin-labeled probes according to (15). the manufacture’s instructions (Boehringer Mannheim GmbH, Mannheim, Germany). A specific probe for MT-III (nucleotide position #173 to #284) was obtained by cleaving rat MTIII cDNA with Mbo II and Nci I. The density of reacted band was measured at 685nm with FluorImager (Molecular Dynamics, Sunnyvale, CA). Levels of mRNA were expressed as a ratio relative to sham-operated rats. Enzyme-linked immunosorbent assay (ELLSA) using phagemid-antibody : MT-III expression ELISA was carried out by the method of Tesar et al (17). In brief, was measured by ELISA. 96-well microtiter plates (Becton Dickison, NJ) were coated with brain homogenates prepared as was used as primary antibody and sheep described (4). The monoclonal phagemid-antibody anti-Ml3 polyclonal affinity purified antibody horseradish peroxidase conjugated was applied as They were stained with o-phenylenediamine sulfate solution and 0.03% H,O, second antibody. in O.lM citric acid and 0.2M Na,HPO,. The color development was measured at 492 nm with an ELISA-reader model Spectra max 250 (Molecular Devices, Sunnyvale, CA). Levels of MTIII were expressed as a ratio relative to sham-operated rats.
Results DNA sequencing of the We raised high affinity phagemid-antibodies specific for rat MT-III. hypervariable region in the high affinity phagemid with rat MT-III was carried out. The major sequence was 5’-cagagggggagggtttagacgcgg-3’, which translated into eight amino acids; Q-R-GWe subcloned this DNA fragment into all of the hypervariable regions in one R-V-S-T-R. phagemid to obtain the monoclonal phagemid-antibody. As shown in Fig. 1, the obtained monoclonal phagemid-antibody reacted with only rat MT-III at the expected position (7.2 kD) By contrast, anti-metallothionein antibody E9 reacted with rat MT-I but not rat MT-I or MT-II. and MT-II, but not MT-III. The time course of MT-III expression after rat transient whole brain ischemia was investigated Four days after ischemic stress, the expression of MT-III in the cerebrum increased by ELISA.
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Induction of MT-III after Ischemia
Vol. 64, No. 8, 1999
to about 2.2-fold that of control @
A
6
kDs B E 4.-32 -
lb
-
7.2 -
1
2
3
1
2
3
Fig. 1 Western blot analysis of MT-I, -11 and -111. 10 ,Y g of MT-I, -11, and -III recombinant proteins were electrophoresed on 15% SDS-polyacrylamide gel, transferred to nitrocellulose membrane and then immunostained with the monoclonal phagemid-antibody (A) or anti-metallothionein antibody E9 (B). Lane 1, MT-I; lane 2, MT-II; lane 3, MT-III.
Induction of MT-III after Ischemia
Vol. 64, No. 8, 1999
A 2.5
*
2
**
1.5 1 0.5iJ@ays)
shemc;
sham
1
,,,,,
2
3
4
7
14 (days)
C 2.5 *
2
*
1.5 1 0.5ifi sham
1
2
3
4
7
14 (days)
Fig. 2 Time course of MT-III protein and mRNA after transient whole brain ischemia. Cerebral protein level (A) and cerebellum protein level (B) was measured by ELISA with the monoclonal phagemid-antibody specific for rat MT-III. Whole brain mRNA level (C) was measured by Northern blot analysis. Levels of protein or mRNA were expressed as a ratio relative to sham-operated rats. Each point represents the mean + SD. P-value against sham control analyzed with Student’s t test. *p
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Fig. 3 Immunohistochemical analysis for MT-III in rat brain. Coronal sections from normal and ischemic injured rat brains were immunostained with the monoclonal phagemid-antibody. Normal cerebrum at low magnification ( X 4) (A) and hippocampus at high magnification (X80) (C), and normal cerebellum at low magnification (X 32) (E) and at high magnification (X 50) (F). (B) and (D) show the same cerebral regions as in (A) and (C) after transient whole brain ischemia. Hi, hippocampus; DG, dentate gyrus; Cx, cortex; T, thalamus; CP, caudate putamen; Am, amygdala; Pl, pyramidal cell layer; Ml, molecular layer; Pu, Purkinje cell layer; Gl, granular layer. Scale bars, lmm (A and B); 50 pm (C, D, E and F).
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Discussion The present study demonstrated that MT-III in normal rat brain is exclusively localized in neurons in hippocampus, dentate gyrus, cerebral cortex, olfactory bulb and Purkinje cells in cerebellum. These results are consistent with previous observations by in situ hybridization studies (6,10,11) that MT-III mRNA is expressed in neuronal cells. The present data also support the observations using mouse MT-III transgenic mice that the MT-III transgene was present in readily identifiable neurons, but not glia (6). In contrast, Tsuji et al. (4,12) reported that the MT-III immunoreactivity was localized most abundantly in the astrocytes of normal human, rat and mouse brain using their own antibodies. The reason for this discrepancy is not clear, but it is possible that their antibodies were not specific for rat MT-III since a rabbit polyclonal antibody against a synthetic polypeptide homologous to the unique four amino acid block near the carboxyl terminus of rat MT-III (4,18) and a monoclonal antibody against human (12), not rat MT-III, were used. Antibodies have problems targeting small epitopes due to steric hindrance by their surface peptides chains. On the other hand, phagemid-antibodies are able to target very small epitopes, because they search for binding sites with only a fingertip rather than two whole hands (17). Rat MT-III has high similarity to rat MT-I and MT-II (66% and 62% identity in amino acid sequence, respectively) (9). Therefore, in this study, we raised a high affinity phagemidantibody for rat MT-III. The phagemid-antibody reacted with only rat MT-III, but not rat MT-I or MT-II, by Western blot analysis (Fig. 1). Immunohistochemical analysis with the phagemidantibody showed that MT-III immunoreactivity is present abundantly in neuronal cell bodies in CAl-3 regions of hippocampus, the dentate gyrus, cerebral cortex, olfactory bulb and Purkinje cells in cerebellum (Fig. 3A,C,E,F). This finding agrees with our observations by in situ hybridization study (unpublished data). The distribution of MT-III shown in this study (Fig. 3A,C,E,F) was similar to that of histochemically reactive zinc as detected with Timm’s stain or 6-methoxy-8-para-toluene sulfonamide quinoline (TSQ) histofluorescence (6). Several studies showed that zinc is neurotoxic (19,20) and its intracellular accumulation may trigger neuronal death caused by seizures (21) and transient cerebral ischemia (22). In addition, MT-III binds zinc in vivo (6) and confers resistance to zinc cytotoxity in cultured cells transfected with MT-III cDNA (23). Erickson et al. (24) reported that MT-III-deficient mice were sensitive to seizure-induced injury to CA3 hippocampal neurons, whereas transgenic mice expressing elevated levels of MT-III were resistant to this damage. Thus, MT-III in hippocampal neurons may have a potential role in detoxification of zinc during neuronal stress. Recently, Yuguchi et al. (10) reported that MT-III mRNA expression increased transiently in neurons of the rat cerebral cortex on day 4 after focal cerebral ischemia. In the present study, ELISA and Northern blot analysis revealed that the expression of MT-III protein as well as mRNA increased in cerebrum on day 4 after ischemic stress (Fig. 2A,C). And immunohistochemical analysis demonstrated intense staining in neurons in injured brain after ischemia, which distributed in the same regions as in normal brain (Fig. 3B,D). After transient cerebral ischemia in rats, zinc accumulated specifically in degenerating neurons in the hippocampus as well as in the cerebral cortex, thalamus, striatum and amygdala (22). We demonstrated that MT-III is up-regulated and expressed at high levels in neurons of cerebrum
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soon after ischemic insult (Fig. 3D). The pattern of the elevated expression of MT-III was similar to that of accumulated zinc. These findings indicate that MT-III is induced only in neurons by ischemic stress and suggest that MT-III is involved in protecting neurons from ischemic insult by reducing the level of neurotoxic zinc. Another specific function of MT-III is growth inhibition of neurons. MT-III inhibits the survival and neurite formation of cortical neurons in vitro (4). High level expression of MT-III in neurons after ischemic insult may prevent neuronal sprouting and inhibit uncontrolled overgrowth of neurites. MT-III levels were reduced in the cerebral cortex of Alzheimer’s disease (AD) brains (4,s). Uchida (25) postulated that a deficit of MT-III in AD brains could lead to excessive stimulation of neurons, resulting in neuronal cell death and that MT-III might be associated with some neurodegenerative disorders. In contrast, Erickson et aZ. (26) reported that neither MT-III mRNA nor protein were significantly decreased in AD brains and that MT-III down-regulation is not an important pathologic event in AD. Therefore, the implication of MT-III in AD pathology is still controversial. The present study demonstrates that MT-III in neurons plays an important role in the central nervous system (CNS) in normal and injured brains. We hypothesize that neurons themselves prevent abortive neuronal sprouting by expressing MT-III to maintain the homeostasis of zinc in the CNS. The specific phagemid-antibody for MT-III raised in this study will be useful to clarify the neurophysiological functions of MT-III in the CNS and neuropathogenic potential in AD.
Acknowledgements We thank Ms. K. Yasaka for excellent technical assistance. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan.
References 1. 2. 3. 4. 5. 6.
7.
J.H.R. dG1 and A. SCdFFER, Biochemistry 27 8509-8515 (1988). R.D. PALMITER, Experientia Suppl. 52 63-80 (1987). M. ASCHNER, M.G. CHERIAN, C.D. KLAASSEN, R.D. PALMITER, J.C. ERICKSON and A.I. BUSH, Toxicol. Appl. Pharmacol. 142 229-242 (1997). Y. UCHIDA, K. TAKIO, K. TITANI, Y. IHARA and M. TOMONAGA, Neuron 7 337-347 (1991). R.D. PALMITER, S.D. FINDLEY, T.E. WHITMORE and D.M. DURMAM, Proc. Natl. Acad. Sci. USA 89 6333-6337 (1992). B.A. MASTERS, C.J. QUAIFE, J.C. ERICKSON, E.J. KELLY, G.J. FROELICK, B.P. ZAMBROWICZ, R.L. BRINSTER and R.D. PALMITER, J. Neurosci. 14 5844-5857 (1994). A.K. SEWELL, L.T. JENSEN, J.C. ERICKSON, R.D. PALMITER and D.R. WINGE, Biochemistry 34 4740-4747 (1995).
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8. 9. 10.
11.
12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.
Induction of MT-III after Ischemia
715
S. TSUJI, H. KOBAYASHI, Y. UCHIDA, Y. IHARA and T. Miyatake, EMBO J. 1148434850 (1992). H. KOBAYASHI, Y. UCHIDA, Y. IHARA, K. NAKAJIMA, S. KOHSAKA, T. MIYATAKE and S. TSUJI, Mol. Brain Res. 19 188-194 (1993). T. YUGUCHI, E. KOHMURA, T. SAKAKI, M. NONAKA, K. YAMADA, T. YAMASHITA, T. KISHIGUCHI, T. SAKAGUCHI and T. HAYAKAWA, J. Cereb. Blood Flow Metab. 17 745-752 (1997). S. CHOUDHURI, K.K. KRAMER, N.E.J. BERMAN, T.P. DALTON, G.K. ANDREWS and C.D. KLAASSEN, Toxicol. Appl. Pharmacol. 131 144-154 (1995). I. HOZUMI, T. INUZUKA, H. ISHIGURO, M. HIRAIWA, Y. UCHIDA and S. TSUJI, Brain Res. 741 197-204 (1996). W.A. PULSINELLI and J.B. BRIERLEY, Stroke 10 267-272 (1979). P. R&ITGEN and J. COLLINS, Gene 164 243-250 (1995). Y. NAKAHASHI, H. FUJITA, S.TAKETANI, N. ISHIDA, A. KAPPAS and S. SASSA, Proc. Natl. Acad. Sci. USA 89 281-285 (1992). B. JASANI and M. E. ELMES, Methods in Enzymology 205 95-107 (1991). M. TESAR, C. BECKMANN, P. RGTTGEN, B. HAASE, U. FAUDE and K.N. TIMMIS, Immunotechnology 153-64 (1995). I. HOZUMI, T. INUZUKA, M. HIRAIWA, Y. UCHIDA, T. ANEZAKI, H. ISHIGURO, H. KOBAYASHI, Y. UDA, T. MIYATAKE and S. TSUJI, Brain Res. 688 143-148 (1995). M. YOKOYAMA, J. KOH and D.W. CHOI, Neurosci. Lett. 71351-355 (1986). D.W. CHOI, M. YOKOYAMA and J. KOH, Neuroscience 24 67-79 (1988). C.J. FREDERICKSON, M.D. HERNANDEZ and J.F. McGINTY, Brain Res. 480 317-321 (1989). J.Y. KOH, S.W. SUH, B.J. GWAG, Y.Y. HE, C.Y. HSU and D.W. CHOI, Science 272 1013-1016 (1996). R.D. PALMITER, Toxicol. Appl. Pharmacol. 135 139-146 (1995). J.C. ERICKSON, G. HOLLOPETER, S.A. THOMAS, G.J. FROELICK and R.D. PALMITER, J. Neurosci. 17 1271-1281(1997). Y. UCHIDA, Biol. Signals 3 211-215 (1994). J.C. ERICKSON, A.K. SEWELL, L.T. JENSEN, D.R. WINGE and R.D. PALMITER, Brain Res. 649 297-304 (1994).