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Adenoviral mediated transfer of TIMP-3 partially prevents glutamate-induced cell death in primary cultured cortical neurons of the rat
Molecular Brain Research 127 (2004) 136 – 139 www.elsevier.com/locate/molbrainres
Short communication
Adenoviral mediated transfer of TIMP-3 partially prevents glutamate-induced cell death in primary cultured cortical neurons of the rat Rongbiao Pi a,1, Wei Yin a,1, Suqiu Zheng a, Pengxin Qiu a, Jian Zhou b, Wei Guo c, Tao Su a, Guangmei Yan a,* a
Department of Pharmacology, Zhongshan Medical College, Sun Yat-Sen University, Guangzhou 510080, PR China b Department of Biotechnology, Institute of Microbiology of Academy of China, Beijing 100800, PR China c Department of Forensic Pathology, Zhongshan Medical College, Sun Yat-Sen University, Guangzhou 510080, PR China Accepted 26 February 2004
Abstract Metalloproteinases and tissue inhibitor of metalloproteinases (TIMPs) are associated with tissue reorganization after injury. The upregulation of TIMP-3 has recently been found in ischemic brain although its functional implications are unclear. In this study we show that overexpression of TIMP-3 by adenovirus-mediated gene transfer partially protects neurons against excitotoxic death induced by glutamate in culture. The partial neuroprotection afforded by TIMP-3 has implications for our understanding of the physiological role of TIMP-3 in the normal and damaged central nervous system. D 2004 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Neuronal death Keywords: Neurons; Adenoviral mediated transfer; TIMP-3; Neuroprotection; Glutamate
Matrix metalloproteinases (MMPs), a large family of endopeptidases, regulate the pericellular environment through the cleavage of membrane receptors, cytokines and protein components of the extracellular matrix. MMPs’ activity is controlled by the multifunctional tissue inhibitors of metalloproteinases. Four members of the TIMP family have been characterized so far, designated as TIMP-1, TIMP-2, TIMP-3, and TIMP-4 [7]. Inhibition of MMPs using synthetic compounds potently attenuated brain ischemic injury, which suggested that MMPs and TIMPs were involved in cell viability and tissue reorganization in the ischemic brain [12]. TIMP-3 is the only member of the TIMP family found exclusively in the extracellular matrix (ECM). Increasing evidence shows that TIMP-3 has addition factions besides
* Corresponding author. Tel.: +86-20-87330230; fax: +86-2087330563. E-mail address: [email protected] (G. Yan). 1 Rongbiao Pi and Wei Yin contributed equally to this manuscript. 0169-328X/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molbrainres.2004.02.026
its MMPs inhibitory activity, such as cell growth promotion [17], matrix binding [5,18], inhibition of angiogenesis [1], proposing cell survival [8] and induction of apoptosis [4,6,14]. In another study, we found that TIMP-3 mRNA was increased in the rat ischemic brain and its protein was increased in primary cultured cortical neurons exposed to toxic glutamate concentration (unpublished data). Transient forebrain ischemia induces selective neuronal death in specific vulnerable areas. This type of neuronal death evolves a few days after transient forebrain ischemia. During that time, many factors contribute to the ischemic injury, such as inflammation. Recently, metalloproteinases were reported to be associated with brain ischemia injury. Knocking out the metalloproteinase 9 gene significantly attenuated ischemic injury in mice and a synthetic inhibitor of metalloproteinases also reduced the infarction size in ischemic rats and mice [2]. Although TIMP-3 has a proapoptotic function in dividing cells [4,6,14], it is unknown whether TIMP-3 can protect postmitotic neurons against death induced by glutamate. In the present study, we used cultured cortical neurons to determine whether adenoviral
R. Pi et al. / Molecular Brain Research 127 (2004) 136–139
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Fig. 1. Detections of TIMP-3 expression following transfection with rAd-gfp-TIMP-3 in primary cultured rat cerebral cortical neurons by immunocytochemistry. Cultured cortical neurons were transfected with adenovirus at DIV 9 and protein was harvested at DIV12. The protein level of TIMP-3 was assayed by immunocytochemistry. A and a: normal; B and b: rAd-gfp; C and c: rAd-gfp-TIMP-3. a, b and c are results under fluorescence microscopy while A, B and C are results of immunocytochemistry. White arrows indicate positive signal. A, B, C, a, b and c: 100.
mediated transfer of TIMP-3 can protect against glutamate toxicity. First, we generated the recombinated adenoviral vector, rAd-gfp-TIMP-3 with the green fluorescence protein (GFP) gene as a reporter by the general strategy previously described [10]. Briefly, the TIMP-3 gene was cloned into the multiple cloning site of the adenoviral shuttle plasmid pAdTrackCMV (kindly provided by Dr. Luo, Sun Yat-Sen University). The resulting plasmid and pAdEasy-1, a plasmid that contains the complete adenovirus 5 genome were cotransfected into the human embryonic kidney cell line 293 (HEK 293) by the lipofectamine transfection method. Infectious viral particles containing the inserted TIMP-3 were generated by in vivo recombination in the HEK293 cells and were isolated as single plaques 10 –20 days later. In addition, a control adenoviral vector without TIMP-3 was also prepared. The isolated plaques were propagated in HEK293 cells for several passages to obtain high titer stocks. Viral particles were purified by CsCl ultracentrifugation. The titer of viral stocks was determined by plaque assay, as high as 1.2 1012 and the viruses were prepared to infect cultured cortical neurons. Primary cortical neuronal cultures were prepared from embryonic 17 – 18 days (E17-18) fetal Sprague – Dawley rats. Cortical cells were plated at a density of 1 106 cells/well in 10% FBS neurobasic medium (NBM) in 12well plates which had previously been coated with poly-Llysine. Cultures were incubated at 37 jC in a humidified incubator with 5% CO2 and 95% air. Cytosine arabinoside (Ara C) at a final concentration of 5 AM was added at around 24 h after plating to inhibit non-neuronal cell division. Two days subsequent to the addition of Ara C, the Ara C-containing medium was replaced by NBM medium containing 2% B27. Subsequent media replacement was carried out twice a week. Experiments were performed on cultures 10 – 12 days after initial plating. The concentration of virus was chosen in order to obtain 95% or better infection. Although there was a low level of
toxicity due to the viral infection, using 10-fold less virus did not alter the amount of cell death among infected neurons, yet the percentage of infected cells decreased to 70%. One day after infection (18 –24 h), the virus was removed and cells were fed with fresh serum-free NBMcontaining B27 media. Three days after infection, cells were photographed using a fluorescence microscope to monitor the reporter gene, GFP and the protein of TIMP-3 was detected by immunocytochemistry and Western blot. For immunocytochemistry, neuronal cultures were rinsed in PBS, fixed in 4% PFA. After that, neurons or sections were incubated with the primary antibody against TIMP-3 for 120 min at room temperature and incubated for another 20 min with the second antibody. Neurons were coated with the label for 20 min and after rinsing in PBS finally incubated with DAB (3,3V-diaminobenzidine)-chromogen for 5 min at
Fig. 2. Over-expression of TIMP-3 in primary cultured rat cerebral cortical neurons by adenoviral vector. Cultured cortical neurons were transfected with adenovirus rAd-gfp-TIMP-3 at DIV 9 and protein was harvested at DIV12. The protein level of TIMP-3 was assayed by Western blot. (A) is a represent result of triplicate experiment and (B) is the quantitative analysis’ results. 1, non-transfected group; 2, transfected group. The statistical analysis was carried out with a Student’s t test. **P < 0.01. n = 3.
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R. Pi et al. / Molecular Brain Research 127 (2004) 136–139
Fig. 3. Overexpression of TIMP-3 attenuated glutamate-induced cell death in primary cultured rat cerebral cortical neurons. Cultured cortical neurons were transfected with adenovirus or not at DIV 9 and then treated with glutamate or not at DIV12. Neuronal viability was assayed 24 h later. *P < 0.05 compared to glutamate-treated group. The statistical analysis was carried out with a Student’s t test, and results were interpreted to be significantly different when P < 0.05. n = 3.
room temperature and then washed with distilled water. Counterstaining was performed with hematoxylin before mounting for Western blotting, cells were harvested in cell lysis buffer [containing (in mM) 20 Tris, pH 7.5, 150 NaCl, 1 EDTA, 1 EGTA, 2.5 sodium pyrophosphate, 1 Na3VO4, and 1 PMSF plus 1% Triton X-100, 1 Ag/ml leupeptin]. After incubation on ice for 15 min and centrifugation at 18,000 g at 4 jC for 10 min, whole protein concentrations were determined by the BCA assay (Pierce, Rockford, IL), using bovine serum albumin as a standard. Cell lysates were diluted in SDS sample buffer (final concentrations: 62.5 mM Tris – HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM DTT, 0.1% bromphenol blue), and the mixture was boiled for 5 min. Protein (30 Ag) was separated on a 10% SDS-poly-
acrylamide gel. Blocking was performed onto polyvinyldifluoride membranes with blocking buffer (20 mM Tris, 136 mM NaCl, pH 7.6, 0.1% Tween 20, 5% nonfat dry milk), and detected by using primary polyclonal antibodies against TIMP-3 (diluted 1:1000; Santa Cruz Biotechnology). After incubation overnight at 4 jC the signals were obtained by binding of a secondary anti-rabbit HRP-linked antibody and were visualized on X-ray films (Hyperfilm, Amersham Biosciences), using a chemiluminescence kit (New England Biolabs). Quantitative analysis of protein levels was performed by densitometric scanning of the autoradiograms with the Bio-Rad quantity one software, and each protein blot is representative of three independent experiments. Transfected with adenovirus for 3 days, cultured cerebral cortical neurons expressed GFP (see Fig. 1b and c). To establish the ideal tranfection rate, neurons were transfected with adenovirus in different concentration. We found the transfection rate was up to 90% when the ratio of virus to neurons is 2000 (data not shown). Potent over-expression of TIMP-3 protein was only observed in cultures transfected with rAd-gfp-TIMP-3 (see Figs. 1C and 2). Neurons were cultured on 96-well plates. After transfection with adenovirus for 3 days, neuorns that had been cultured for 12 days in vitro were rinsed three times in NBM containing 2% B27 supplemented with glucose and penicillin/streptomycin and then were maintained in the same medium. Control cultures were treated identically but were maintained in normal medium and then were added glutamate (200 AM) directly into the medium for 24 h to induce neurotoxicity. Neurotoxicity was assessed using the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye (MTT) assay [15]. Fig. 3 showed that overexpression of TIMP-3 protein partially blocked glutamate-induced neurotoxicity.
Fig. 4. The glutamate-induced nuclear condensation of neurons was blocked by overexpression TIMP-3 in primary cultured rat cerebral cortical neurons. Before exposure to 200 AM glutamate, neurons were transfected with rAd-gfp-TIMP-3 or rAd-gfp or not (Adenoviral particles/neurons, 2000/1) for three days. After 24 h, neurons were fixed with 4% para-formaldehyde and then stained with Hoechst33258. Condensed neuronal nuclei were observed under fluorescent microscopy. (A) Glutamate treated normal neurons; (B) Glutamte treated rAd-gfp infected neurons; (C) Glutamte treated rAd-gfp-TIMP-3 infected neurons; (D) Glutamate untreated neurons (control). ##P < 0.01, compared to groups D; *P < 0.05, compared to groups A and B respectively. The statistical analysis was carried out with a Student’s t-test, and results were interpreted to be significantly different when P < 0.05. n = 3.
R. Pi et al. / Molecular Brain Research 127 (2004) 136–139
Glutamate induces apoptosis and necrosis in neurons. To determine whether the overexpression of TIMP-3 protein blocked glutamate-induced nuclear condensation, a typically apoptotic character, Hoechst 33258 nuclear staining was carried out. Neurons were fixed in 4% formalin for 30 min. After two 5-min washes in PBS, neurons were incubated with Hoechst 33258 (1 Ag/ml) at room temperature for 20 min. Images of microscopic fields were captured. The proportion of apoptotic cells was estimated using a graticule eyepiece and counting approximately 350 cells per dish. Fig. 4 showed that overexpression of TIMP-3 protein decreased the number of condensed neuronal nuclei, a typical characteristic of apoptosis. TIMP-3 was first cloned by Blenis et al. in the extracellular matrix (ECM) of chicken embryo fibroblasts and it is the only member that is tightly bound to ECM [5]. Recently, TIMP-3 has been found to exhibit several biochemical and physiological/biological functions, including inhibition of active MMPs [9], cell growth promotion [17], matrix binding [5,18], inhibition of angiogenesis [6], proposing cell survival [8] and induction of apoptosis [4,6,14]. Mutations in TIMP-3 are the cause of Sorsby’s fundus dystrophy in humans, a disease that results in early onset macular degeneration [14] and accelerated apoptosis was found in the TIMP-3-deficient mammary gland [8]. These findings suggested that TIMP-3 might be a factor involved in cell survival in certain tissues or cells. Most recently, Wallace et al. [16] considered that TIMP-3 might be a proapoptotic factor but they had no direct evidence. In our study, overexpression of TIMP-3 was observed to partially block glutamate-induced neurotoxicity in primary cultured cortical neurons. Hoechst 33258 staining showed TIMP-3 blocked apoptosis induced by glutamate. Although we did not determine the mechanisms conferring TIMP-3 neuroprotective function, our findings suggested that TIMP-3 might be a protective factor enhancing neuronal survival in ischemic brain. PI-3 Kinase/Akt pathway are very important survival pathway in many kinds of cells [3,11]. Recently, TIMP-1 was reported to protect breast epithelial cells against intrinsic apoptotic cell death via the FAK/PI 3-kinase and MAPK signaling pathway [13]. TIMP-3 might also activate the survival pathway to protect neurons against glutamateinduced apoptosis. In conclusion, our findings demonstrate that overexprssion of TIMP-3 partially blocks the apoptosis induced by glutamate. Further experiments may uncover the protective mechanisms of TIMP-3 and may point to the mechanisms of some diseases, such as brain ischemia and Sorsby’s fundus dystrophy.
Acknowledgements This research was supported by National Outstanding Youth Foundation of Nature Scientific (Nv: 960125) and America-Sino Medical Foundation.
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References [1] B. Anand-Apte, M.S. Pepper, E. Voest, R. Montesano, B. Olsen, G. Murphy, S.S. Apte, B. Zetter, Inhibition of angiogenesis by tissue inhibitor of metalloproteinase-3, Investig. Ophthalmol. Vis. Sci. 38 (1997) 817 – 823. [2] M. Asahi, X. Wang, T. Mori, T. Sumii, J.C. Jung, M.A. Moskowitz, M.E. Fini, E.H. Lo, Effects of matrix metalloproteinase-9 gene knock-out on the proteolysis of blood-brain barrier and white matter components after cerebral ischemia, J. Neurosci. 21 (2001) 7724 – 7732. [3] B.A. Ballif, J. Blenis, Molecular mechanisms mediating mammalian mitogen-activated protein kinase (MAPK) kinase (MEK)MAPK cell survival signals, Cell Growth Differ. 12 (2001) 397 – 408. [4] J. Bian, Y. Wang, M.R. Smith, H. Kim, C. Jacobs, J. Jackman, H.F. Kung, N.H. Colburn, Y. Sun, Suppression of in vivo tumor growth and induction of suspension cell death by tissue inhibitor of metalloproteinases (TIMP)-3, Carcinogenesis 17 (1996) 1805 – 1811. [5] J. Blenis, S.P. Hawkes, Characterization of a transformation-sensitive protein in the extracellular matrix of chicken embryo fibroblasts, J. Biol. Chem. 259 (1984) 11563 – 11570. [6] M. Bond, G. Murphy, M.R. Bennett, A.C. Newby, A.H. Baker, Tissue inhibitor of metalloproteinase-3 induces a Fas-associated death domain-dependent type II apoptotic pathway, J. Biol. Chem. 277 (2002) 13787 – 13795. [7] K. Brew, D. Dinakarpandian, H. Nagase, Tissue inhibitors of metalloproteinases: evolution, structure and function, Biochim. Biophys. Acta 1477 (2000) 267 – 283. [8] J.E. Fata, K.J. Leco, E.B. Voura, H.Y. Yu, P. Waterhouse, G. Murphy, R.A. Moorehead, R. Khokha, Accelerated apoptosis in the Timp-3deficient mammary gland, J. Clin. Invest. 108 (2001) 831 – 841. [9] D.E. Gomez, D.F. Alonso, H. Yoshiji, U.P. Thorgeirsson, Tissue inhibitors of metalloproteinases: structure, regulation and biological functions, Eur. J. Cell Biol. 74 (1997) 111 – 122. [10] T.C. He, S. Zhou, L.T. da Costa, J. Yu, K.W. Kinzler, B. Vogelstein, A simplified system for generating recombinant adenoviruses, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 2509 – 2514. [11] B.A. Hemmings, Akt signaling: linking membrane events to life and death decisions, Science 275 (1997) 628 – 630. [12] X. Jiang, S. Namura, I. Nagata, Matrix metalloproteinase inhibitor KB-R7785 attenuates brain damage resulting from permanent focal cerebral ischemia in mice, Neurosci. Lett. 305 (2001) 41 – 44. [13] X.W. Liu, M.M. Bernardo, R. Fridman, H.R. Kim, Tissue inhibitor of metalloproteinase-1 (TIMP-1) protects human breast epithelial cells against intrinsic apoptotic cell death via the FAK/PI 3-kinase and MAPK signaling pathway, J. Biol. Chem. 41 (2003) 40364 – 40372. [14] M.A. Majid, V.A. Smith, D.L. Easty, A.H. Baker, A.C. Newby, Sorsby’s fundus dystrophy mutant tissue inhibitors of metalloproteinase-3 induce apoptosis of retinal pigment epithelial and MCF-7 cells, FEBS Lett. 529 (2002) 281 – 285. [15] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1983) 55 – 63. [16] J.A. Wallace, S. Alexander, E.Y. Estrada, C. Hines, L.A. Cunningham, G.A. Rosenberg, Tissue inhibitor of metalloproteinase-3 is associated with neuronal death in reperfusion injury, J. Cereb. Blood Flow Metab. 22 (2002) 1303 – 1310. [17] T.T. Yang, S.P. Hawkes, Role of the 21-kDa protein TIMP-3 in oncogenic transformation of cultured chicken embryo fibroblasts, Proc. Natl. Acad. Sci. U. S. A. 89 (1992) 10676 – 10680. [18] W.H. Yu, S. Yu, Q. Meng, K. Brew , J.F. Woessner Jr., TIMP-3 binds to sulfated glycosaminoglycans of the extracellular matrix, J. Biol. Chem. 275 (2000) 31226 – 31232.
Short communication
Adenoviral mediated transfer of TIMP-3 partially prevents glutamate-induced cell death in primary cultured cortical neurons of the rat Rongbiao Pi a,1, Wei Yin a,1, Suqiu Zheng a, Pengxin Qiu a, Jian Zhou b, Wei Guo c, Tao Su a, Guangmei Yan a,* a
Department of Pharmacology, Zhongshan Medical College, Sun Yat-Sen University, Guangzhou 510080, PR China b Department of Biotechnology, Institute of Microbiology of Academy of China, Beijing 100800, PR China c Department of Forensic Pathology, Zhongshan Medical College, Sun Yat-Sen University, Guangzhou 510080, PR China Accepted 26 February 2004
Abstract Metalloproteinases and tissue inhibitor of metalloproteinases (TIMPs) are associated with tissue reorganization after injury. The upregulation of TIMP-3 has recently been found in ischemic brain although its functional implications are unclear. In this study we show that overexpression of TIMP-3 by adenovirus-mediated gene transfer partially protects neurons against excitotoxic death induced by glutamate in culture. The partial neuroprotection afforded by TIMP-3 has implications for our understanding of the physiological role of TIMP-3 in the normal and damaged central nervous system. D 2004 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Neuronal death Keywords: Neurons; Adenoviral mediated transfer; TIMP-3; Neuroprotection; Glutamate
Matrix metalloproteinases (MMPs), a large family of endopeptidases, regulate the pericellular environment through the cleavage of membrane receptors, cytokines and protein components of the extracellular matrix. MMPs’ activity is controlled by the multifunctional tissue inhibitors of metalloproteinases. Four members of the TIMP family have been characterized so far, designated as TIMP-1, TIMP-2, TIMP-3, and TIMP-4 [7]. Inhibition of MMPs using synthetic compounds potently attenuated brain ischemic injury, which suggested that MMPs and TIMPs were involved in cell viability and tissue reorganization in the ischemic brain [12]. TIMP-3 is the only member of the TIMP family found exclusively in the extracellular matrix (ECM). Increasing evidence shows that TIMP-3 has addition factions besides
* Corresponding author. Tel.: +86-20-87330230; fax: +86-2087330563. E-mail address: [email protected] (G. Yan). 1 Rongbiao Pi and Wei Yin contributed equally to this manuscript. 0169-328X/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molbrainres.2004.02.026
its MMPs inhibitory activity, such as cell growth promotion [17], matrix binding [5,18], inhibition of angiogenesis [1], proposing cell survival [8] and induction of apoptosis [4,6,14]. In another study, we found that TIMP-3 mRNA was increased in the rat ischemic brain and its protein was increased in primary cultured cortical neurons exposed to toxic glutamate concentration (unpublished data). Transient forebrain ischemia induces selective neuronal death in specific vulnerable areas. This type of neuronal death evolves a few days after transient forebrain ischemia. During that time, many factors contribute to the ischemic injury, such as inflammation. Recently, metalloproteinases were reported to be associated with brain ischemia injury. Knocking out the metalloproteinase 9 gene significantly attenuated ischemic injury in mice and a synthetic inhibitor of metalloproteinases also reduced the infarction size in ischemic rats and mice [2]. Although TIMP-3 has a proapoptotic function in dividing cells [4,6,14], it is unknown whether TIMP-3 can protect postmitotic neurons against death induced by glutamate. In the present study, we used cultured cortical neurons to determine whether adenoviral
R. Pi et al. / Molecular Brain Research 127 (2004) 136–139
137
Fig. 1. Detections of TIMP-3 expression following transfection with rAd-gfp-TIMP-3 in primary cultured rat cerebral cortical neurons by immunocytochemistry. Cultured cortical neurons were transfected with adenovirus at DIV 9 and protein was harvested at DIV12. The protein level of TIMP-3 was assayed by immunocytochemistry. A and a: normal; B and b: rAd-gfp; C and c: rAd-gfp-TIMP-3. a, b and c are results under fluorescence microscopy while A, B and C are results of immunocytochemistry. White arrows indicate positive signal. A, B, C, a, b and c: 100.
mediated transfer of TIMP-3 can protect against glutamate toxicity. First, we generated the recombinated adenoviral vector, rAd-gfp-TIMP-3 with the green fluorescence protein (GFP) gene as a reporter by the general strategy previously described [10]. Briefly, the TIMP-3 gene was cloned into the multiple cloning site of the adenoviral shuttle plasmid pAdTrackCMV (kindly provided by Dr. Luo, Sun Yat-Sen University). The resulting plasmid and pAdEasy-1, a plasmid that contains the complete adenovirus 5 genome were cotransfected into the human embryonic kidney cell line 293 (HEK 293) by the lipofectamine transfection method. Infectious viral particles containing the inserted TIMP-3 were generated by in vivo recombination in the HEK293 cells and were isolated as single plaques 10 –20 days later. In addition, a control adenoviral vector without TIMP-3 was also prepared. The isolated plaques were propagated in HEK293 cells for several passages to obtain high titer stocks. Viral particles were purified by CsCl ultracentrifugation. The titer of viral stocks was determined by plaque assay, as high as 1.2 1012 and the viruses were prepared to infect cultured cortical neurons. Primary cortical neuronal cultures were prepared from embryonic 17 – 18 days (E17-18) fetal Sprague – Dawley rats. Cortical cells were plated at a density of 1 106 cells/well in 10% FBS neurobasic medium (NBM) in 12well plates which had previously been coated with poly-Llysine. Cultures were incubated at 37 jC in a humidified incubator with 5% CO2 and 95% air. Cytosine arabinoside (Ara C) at a final concentration of 5 AM was added at around 24 h after plating to inhibit non-neuronal cell division. Two days subsequent to the addition of Ara C, the Ara C-containing medium was replaced by NBM medium containing 2% B27. Subsequent media replacement was carried out twice a week. Experiments were performed on cultures 10 – 12 days after initial plating. The concentration of virus was chosen in order to obtain 95% or better infection. Although there was a low level of
toxicity due to the viral infection, using 10-fold less virus did not alter the amount of cell death among infected neurons, yet the percentage of infected cells decreased to 70%. One day after infection (18 –24 h), the virus was removed and cells were fed with fresh serum-free NBMcontaining B27 media. Three days after infection, cells were photographed using a fluorescence microscope to monitor the reporter gene, GFP and the protein of TIMP-3 was detected by immunocytochemistry and Western blot. For immunocytochemistry, neuronal cultures were rinsed in PBS, fixed in 4% PFA. After that, neurons or sections were incubated with the primary antibody against TIMP-3 for 120 min at room temperature and incubated for another 20 min with the second antibody. Neurons were coated with the label for 20 min and after rinsing in PBS finally incubated with DAB (3,3V-diaminobenzidine)-chromogen for 5 min at
Fig. 2. Over-expression of TIMP-3 in primary cultured rat cerebral cortical neurons by adenoviral vector. Cultured cortical neurons were transfected with adenovirus rAd-gfp-TIMP-3 at DIV 9 and protein was harvested at DIV12. The protein level of TIMP-3 was assayed by Western blot. (A) is a represent result of triplicate experiment and (B) is the quantitative analysis’ results. 1, non-transfected group; 2, transfected group. The statistical analysis was carried out with a Student’s t test. **P < 0.01. n = 3.
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R. Pi et al. / Molecular Brain Research 127 (2004) 136–139
Fig. 3. Overexpression of TIMP-3 attenuated glutamate-induced cell death in primary cultured rat cerebral cortical neurons. Cultured cortical neurons were transfected with adenovirus or not at DIV 9 and then treated with glutamate or not at DIV12. Neuronal viability was assayed 24 h later. *P < 0.05 compared to glutamate-treated group. The statistical analysis was carried out with a Student’s t test, and results were interpreted to be significantly different when P < 0.05. n = 3.
room temperature and then washed with distilled water. Counterstaining was performed with hematoxylin before mounting for Western blotting, cells were harvested in cell lysis buffer [containing (in mM) 20 Tris, pH 7.5, 150 NaCl, 1 EDTA, 1 EGTA, 2.5 sodium pyrophosphate, 1 Na3VO4, and 1 PMSF plus 1% Triton X-100, 1 Ag/ml leupeptin]. After incubation on ice for 15 min and centrifugation at 18,000 g at 4 jC for 10 min, whole protein concentrations were determined by the BCA assay (Pierce, Rockford, IL), using bovine serum albumin as a standard. Cell lysates were diluted in SDS sample buffer (final concentrations: 62.5 mM Tris – HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM DTT, 0.1% bromphenol blue), and the mixture was boiled for 5 min. Protein (30 Ag) was separated on a 10% SDS-poly-
acrylamide gel. Blocking was performed onto polyvinyldifluoride membranes with blocking buffer (20 mM Tris, 136 mM NaCl, pH 7.6, 0.1% Tween 20, 5% nonfat dry milk), and detected by using primary polyclonal antibodies against TIMP-3 (diluted 1:1000; Santa Cruz Biotechnology). After incubation overnight at 4 jC the signals were obtained by binding of a secondary anti-rabbit HRP-linked antibody and were visualized on X-ray films (Hyperfilm, Amersham Biosciences), using a chemiluminescence kit (New England Biolabs). Quantitative analysis of protein levels was performed by densitometric scanning of the autoradiograms with the Bio-Rad quantity one software, and each protein blot is representative of three independent experiments. Transfected with adenovirus for 3 days, cultured cerebral cortical neurons expressed GFP (see Fig. 1b and c). To establish the ideal tranfection rate, neurons were transfected with adenovirus in different concentration. We found the transfection rate was up to 90% when the ratio of virus to neurons is 2000 (data not shown). Potent over-expression of TIMP-3 protein was only observed in cultures transfected with rAd-gfp-TIMP-3 (see Figs. 1C and 2). Neurons were cultured on 96-well plates. After transfection with adenovirus for 3 days, neuorns that had been cultured for 12 days in vitro were rinsed three times in NBM containing 2% B27 supplemented with glucose and penicillin/streptomycin and then were maintained in the same medium. Control cultures were treated identically but were maintained in normal medium and then were added glutamate (200 AM) directly into the medium for 24 h to induce neurotoxicity. Neurotoxicity was assessed using the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye (MTT) assay [15]. Fig. 3 showed that overexpression of TIMP-3 protein partially blocked glutamate-induced neurotoxicity.
Fig. 4. The glutamate-induced nuclear condensation of neurons was blocked by overexpression TIMP-3 in primary cultured rat cerebral cortical neurons. Before exposure to 200 AM glutamate, neurons were transfected with rAd-gfp-TIMP-3 or rAd-gfp or not (Adenoviral particles/neurons, 2000/1) for three days. After 24 h, neurons were fixed with 4% para-formaldehyde and then stained with Hoechst33258. Condensed neuronal nuclei were observed under fluorescent microscopy. (A) Glutamate treated normal neurons; (B) Glutamte treated rAd-gfp infected neurons; (C) Glutamte treated rAd-gfp-TIMP-3 infected neurons; (D) Glutamate untreated neurons (control). ##P < 0.01, compared to groups D; *P < 0.05, compared to groups A and B respectively. The statistical analysis was carried out with a Student’s t-test, and results were interpreted to be significantly different when P < 0.05. n = 3.
R. Pi et al. / Molecular Brain Research 127 (2004) 136–139
Glutamate induces apoptosis and necrosis in neurons. To determine whether the overexpression of TIMP-3 protein blocked glutamate-induced nuclear condensation, a typically apoptotic character, Hoechst 33258 nuclear staining was carried out. Neurons were fixed in 4% formalin for 30 min. After two 5-min washes in PBS, neurons were incubated with Hoechst 33258 (1 Ag/ml) at room temperature for 20 min. Images of microscopic fields were captured. The proportion of apoptotic cells was estimated using a graticule eyepiece and counting approximately 350 cells per dish. Fig. 4 showed that overexpression of TIMP-3 protein decreased the number of condensed neuronal nuclei, a typical characteristic of apoptosis. TIMP-3 was first cloned by Blenis et al. in the extracellular matrix (ECM) of chicken embryo fibroblasts and it is the only member that is tightly bound to ECM [5]. Recently, TIMP-3 has been found to exhibit several biochemical and physiological/biological functions, including inhibition of active MMPs [9], cell growth promotion [17], matrix binding [5,18], inhibition of angiogenesis [6], proposing cell survival [8] and induction of apoptosis [4,6,14]. Mutations in TIMP-3 are the cause of Sorsby’s fundus dystrophy in humans, a disease that results in early onset macular degeneration [14] and accelerated apoptosis was found in the TIMP-3-deficient mammary gland [8]. These findings suggested that TIMP-3 might be a factor involved in cell survival in certain tissues or cells. Most recently, Wallace et al. [16] considered that TIMP-3 might be a proapoptotic factor but they had no direct evidence. In our study, overexpression of TIMP-3 was observed to partially block glutamate-induced neurotoxicity in primary cultured cortical neurons. Hoechst 33258 staining showed TIMP-3 blocked apoptosis induced by glutamate. Although we did not determine the mechanisms conferring TIMP-3 neuroprotective function, our findings suggested that TIMP-3 might be a protective factor enhancing neuronal survival in ischemic brain. PI-3 Kinase/Akt pathway are very important survival pathway in many kinds of cells [3,11]. Recently, TIMP-1 was reported to protect breast epithelial cells against intrinsic apoptotic cell death via the FAK/PI 3-kinase and MAPK signaling pathway [13]. TIMP-3 might also activate the survival pathway to protect neurons against glutamateinduced apoptosis. In conclusion, our findings demonstrate that overexprssion of TIMP-3 partially blocks the apoptosis induced by glutamate. Further experiments may uncover the protective mechanisms of TIMP-3 and may point to the mechanisms of some diseases, such as brain ischemia and Sorsby’s fundus dystrophy.
Acknowledgements This research was supported by National Outstanding Youth Foundation of Nature Scientific (Nv: 960125) and America-Sino Medical Foundation.
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