- Email: [email protected]
HSP70 protects murine astrocytes from glucose deprivation injury
Neuroscience Letters 224 (1997) 9–12
HSP70 protects murine astrocytes from glucose deprivation injury Lijun Xu, Rona G. Giffard* Department of Anesthesia, S272, Stanford University School of Medicine, Stanford, CA 94305-5117, USA Received 22 November 1996; revised version received 31 January 1997; accepted 3 February 1997
Abstract Expression of the 70 kDa heat shock protein (HSP70) induced by a first insult is associated with protection from a subsequent ischemic insult in brain. Expression of the human inducible HSP70 was previously shown to protect astrocytes in primary culture from combined oxygen-glucose deprivation. These studies have now been extended to demonstrate that HSP70 expression also protects from isolated glucose deprivation. Slight protection was seen against hydrogen peroxide (H2O2) exposure. Glutathione levels decrease less after glucose deprivation or H2O2 exposure (200 mM) in the cells overexpressing HSP70, compared to either b-galactosidase expressing or uninfected controls (P , 0.01). These data suggest that the HSP70-expressing cells suffered less oxidative stress since their glutathione levels were better preserved. 1997 Elsevier Science Ireland Ltd. Keywords: Retrovirus; Glutathione; Hydrogen peroxide; Mouse; Hypoglycemia; Primary culture; Oxidative stress; Heat shock proteins
Heat shock proteins, and the inducible member of the approximately 70 kDa family, HSP70, in particular, have attracted interest as potential neuroprotectants because prior induction is associated with protection of cultured cells against several different injuries [1,2,11,15,16,20]. Brief pretreatment with ischemia has been shown to protect against both global [9] and focal ischemia [5] and is associated with induction of the heat shock protein HSP70. We have previously reported that expressing human inducible HSP70 using a retroviral vector protects astrocytes in primary culture from combined oxygen-glucose deprivation or a hyperthermic insult [15]. In this paper, we have investigated the range of insults HSP70 expression can protect against by testing glucose deprivation, and hydrogen peroxide (H2O2) exposure. Each represents one aspect of the injury caused by ischemia. While H2O2 is a pure oxidative insult, glucose deprivation represents both an oxidative stress and the loss of substrate. Glutathione (GSH) plays an important role in protecting cells from oxidative stress and in free-radical scavenging [17]. For this reason, we determined the effect of HSP70 overexpression on the decrease in GSH levels caused by these two insults. * Corresponding author. Tel.: +1 415 7258482; fax: +1 415 7258052; e-mail: [email protected]
Primary astrocyte cultures were prepared from postnatal (days 1–3) Swiss Webster mice (Simonsen, Gilroy, CA, USA) as previously described [7]. All procedures were carried out according to a protocol approved by the Stanford University animal care and use committee and were in keeping with the NIH guide. In brief, animals were anesthetized with halothane, decapitated, and the neocortical tissue was freed of meninges and blood vessels, before being minced and suspended in Eagle’s minimal essential medium (MEM; Gibco, Grand Island, NY, USA) supplemented with 21 mM glucose and containing 0.09% trypsin for 20 min at 37°C. The dissociated cells were washed, triturated and plated in 15 mm Falcon Primaria 24-well plates (Becton Dickinson, Lincoln, IL, USA) at a density of 1.8 hemispheres/multiwell (about 2 × 105 cells/cm2) in plating medium consisting of MEM supplemented with 10% equine serum (Hyclone, Logan, UT, USA), 10% fetal bovine serum (Hyclone), 21 mM glucose (Sigma, St. Louis, MO, USA) and 10 ng/ml epidermal growth factor (Sigma). Astrocytes were maintained in a 37°C humidified incubator with a 5% CO2 in room air atmosphere. Immunocytochemical studies revealed that more than 95% of the cells in our cultures stained for glial fibrillary acidic protein. Retroviruses were produced in W-2 packaging cells and
0304-3940/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940 (97 )1 3444-9
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L. Xu, R.G. Giffard / Neuroscience Letters 224 (1997) 9–12
used as previously described [15]. Control retrovirus MPZen-lacZ expressing b-galactosidase from the 5′ LTR promoter and neomycin resistance from an SV40 promoter was the kind gift of Dr. David Vaux. Packaging cells producing pMV6-hsp-70 [11], to express the human inducible HSP70 was the kind gift of Dr. Nahid Mivechi. Cultures were infected 36–48 h after plating by exposure to medium from the packaging cells (after 0.2 mm filtration) and containing 8 mg/ml polybrene (Sigma). Infections were repeated twice. Cells expressing the viral genes were selected in 1 mg/ml G418 for 5 days. All uninfected astrocytes died after 5 days of exposure to G418. The infected astrocytes became essentially confluent within 2 weeks, during which time they were fed twice weekly with plating medium containing 100 mg/ml G418. Cells were used for experiments between days 25–45 in vitro. Expression of HSP70 was confirmed by immunohistochemical staining and immunoblot as previously described [15]. Both cells infected with a retrovirus directing expression of b-galactosidase and uninfected astrocytes, which were not exposed to G418 selection, were used as controls. Experiments were performed at least three times on cultures from a minimum of three different dissections. Astrocyte injury was estimated morphologically by phase-contrast light microscopy and quantitated by measuring lactate dehydrogenase (LDH) activity released from lysed cells into the medium [10]. Total LDH release corresponding to complete astrocyte death was determined at the end of each experiment following freezing at −70°C and rapid thawing. GSH was measured before and after glucose deprivation (GD) for 10 h as described by Ferna´ndez-Checa and Kaplowitz [8] with several modifications. Astrocyte cultures were washed twice with phosphate-buffered saline (PBS), incubated at 37°C for 10 min with 50 mM monochlorobimane (Molecular Probes, Eugene, OR, USA), then washed with PBS and lysed in 0.2% Triton X-100. The insoluble debris was sedimented at 3000 × g for 5 min. The concentration of protein in the supernatant was determined using the bicinchoninic acid method (Pierce, Rockford, IL, USA). The fluorescence of monochlorobimane in the supernatant was measured at 400 nm excitation, 480 nm emission in a Perkin Elmer LS50B spectrofluorimeter. The concentration of GSH was calculated from standard curves and expressed as nmol/mg protein. The relationship between GSH and fluorescence at 480 nm was linear in the range found in the cells. The GSH measurements were also performed after 1 h exposure to 200 mM H2O2, an insult resulting in little cell death. Cultures were deprived of glucose by replacing the culture medium with balanced salt solution (BSS0), at pH 7.4, containing (in mM) NaCl 116, CaCl2 1.8, MgSO4 0.8, KCl 5.4, NaH2PO4 1, NaHCO3 14.7, N-(2-hydroxyethyl)piperazine-N′-(ethanesulfonic acid) (HEPES) 10; and phenol red 10 mg/l lacking glucose. The medium was replaced in each well three times with 800 ml BSS0 resulting in a greater than
2000-fold dilution of growth medium. The medium was sampled for LDH after 10, 18, and 24 h of GD. Astrocyte cultures had their medium exchanged three times with balanced salt solution containing glucose (BSS5.5; BSS0 containing 5.5 mM glucose) at pH 7.4. H2O2 (30%; Sigma) was diluted in the same solution and added to the wells to achieve a final concentration of 200 or 400 mM. The medium was sampled for LDH release after 2, 4, and 6 h of exposure. Values shown are the mean ± SE. Statistical significance (P , 0.05) was determined by ANOVA and Student Neuman–Keuls test for multiple comparisons using SigmaStat (Jandel Scientific). Astrocytes overexpressing HSP70 were significantly more resistant to glucose deprivation than b-galactosidase expressing and uninfected controls, morphologically (Fig. 1) and as assessed by LDH release (Fig. 2A). Astrocytes
Fig. 1. Astrocytes are protected from glucose deprivation (GD) by overexpression of HSP70. Cultures were deprived of glucose by replacing the culture medium with balanced salt solution lacking glucose (BSSo), at pH 7.4. After 24 h, the cells were stained with trypan blue. Pictures were taken at 200 × magnification of the same field with phase contrast (A,C,E) and bright field (B,D,F) optics (bar, 25 mm). HSP70-infected astrocytes were slightly injured and very few cells died (A,B). Cells were almost completely dead and began to peel off in b-galactosidase-expressing cultures (C,D); and uninfected control cultures (E,F).
L. Xu, R.G. Giffard / Neuroscience Letters 224 (1997) 9–12
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overexpressing HSP70 showed no significant protection from 4 or 6 h of 400 mM H2O2 exposure (Fig. 2B), but a small but statistically significant reduction of injury was observed at 2 h. As a measure of the cells’ level of oxidative stress, we then examined the drop in GSH levels after each of the two injury paradigms (Fig. 3). GSH levels were not different among the three groups of astrocytes under normal growth
Fig. 3. GSH was measured before and after 10 h GD or 1 h H2O2 (200 mM) exposure in astrocytes overexpressing HSP70 or b-galactosidase, and uninfected controls. GSH values were standardized to protein. The level of GSH was not different between groups before injury. GSH was significantly decreased in b-galactosidase-expressing and uninfected controls (*P , 0.01), but no significant decrease was evident in the HSP70infected astrocytes after 10 h GD. GSH was significantly higher after GD in HSP70 expressing cultures compared to the b-galactosidase-expressing (*) and uninfected control (#) astrocytes (P , 0.05). All three groups of astrocytes showed significant decreases in GSH after 1 h exposure to H2O2 (P , 0.01) compared to the level in growth medium. However, GSH levels in HSP70 expressing astrocytes were higher than in the other two groups after this insult (P , 0.05, n = 12, mean ± SE).
Fig. 2. (A) Overexpression of HSP70 protects astrocytes from GD. Cultures were washed into balanced salt solution lacking glucose for 24 h. Each point represents the mean of the LDH release (n = 20, mean ± SE) expressed as a percent of maximum LDH release measured after freeze thaw. The HSP70 values are significantly different (P , 0.05) from the bgalactosidase (*) and control (#) values. (B) Effect of HSP70 expression on 400 mM H2O2 injury. Astrocytes which overexpress HSP70, b-galactosidase, and uninfected controls were exposed to 400 mM H2O2 (final concentration in each well) in BSS5.5 for 6 h. LDH release shows that astrocytes are protected by HSP70 expression only at 2 h compared to b-galactosidase-expressing (*) and uninfected control (#) astrocytes (P , 0.05, n = 12, mean ± SE).
conditions. However, after 10 h of glucose deprivation, GSH levels did not significantly decrease in the HSP70 expressing cells while significant drops in both the b-galactosidase (20.1 ± 3.5% decrease) and control astrocytes (30.8 ± 5.4% decrease) were found. GD of 10 h was chosen to measure GSH because at this time the cells were still intact as evidenced by ability to exclude trypan blue, and detection of less than 10% LDH release into the medium. Levels of GSH after exposure to 200 mM H2O2 for 1 h were significantly lower than those in normal growth medium in all three groups. However, the HSP70-expressing cells showed the smallest decrease, 35.6 ± 2.5%, when compared with b-galactosidase expressing cells, 52.8 ± 4.8% decrease, or control uninfected cells, 46.6 ± 3.2% decrease. The difference between groups after H2O2 was significant at P , 0.05. After 1 h exposure to 200 mM H2O2 cell death was less than 10% in HSP70 and bgalactosidase expressing astrocytes, and less than 25% in control astrocytes by both LDH release and trypan blue staining. Both constitutive and inducible members of the HSP70 family of heat shock proteins play a role as molecular chaperones, assisting during protein synthesis and maintaining proteins in a conformation appropriate for transport into subcellular organelles [3,14]. The inducible form of HSP70 is difficult to detect in rat brain in the absence of stressful conditions by immunochemical assay [12,14]. Induction of HSP70 may also be a sensitive marker of
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acute stress such as ischemia [18]. Heat shock protein induction caused by heat or brief ischemia is associated with protection from a second insult in brain [5,6,12]. As we and others previously found, HSP70 is induced in response to heat and combined oxygen-glucose deprivation in cultured murine cortical astrocytes [13]. In this paper, we found that isolated increases in HSP70 expression were sufficient to provide protection against glucose deprivation and, to a minor extent, H2O2 exposure. Both of these insults reflect subsets of the injury that occurs during cerebral ischemia. Since both of these insults involve oxidative stress, we measured glutathione levels and found that cells protected by overexpression of HSP70 were more resistant to the fall in GSH induced by these injuries. After 10 h glucose deprivation HSP70expressing cells did not show any decrease in GSH, whereas GSH in the b-galactosidase expressing and control astrocytes dropped significantly. Similarly, after H2O2 exposure the HSP70 expressing cells showed the smallest decrease. No difference was evident in levels of GSH in the three groups of astrocytes under normal growth conditions. Our findings demonstrate a negative relationship between the level of GSH and injury. Decreased injury by glucose deprivation or H2O2 exposure was observed in concert with higher GSH levels. One possible explanation of this observation is that overexpression of HSP70 leads to decreased demand for new protein synthesis in response to stress. This can lead to better maintenance of amino acid precursor pools and ATP, both of which are needed for GSH synthesis. Higher levels of GSH synthesis could lead to protection from oxidative insults. During glucose deprivation or H2O2 exposure, cells are subjected to oxidative stress sufficient to be cytotoxic. Reactive oxygen species are also important mediators of hypoxic-ischemic central nervous system injury [4,19]. Our results show HSP70 overexpression in astrocytes is sufficient to protect from glucose deprivation and allows maintenance of higher GSH levels. This work was supported in part by NIH grant GM 49831 to R.G.G. The authors wish to thank Dr. David Vaux for the kind gift of packaging cells producing MPZen-lacZ, and Dr. Nahid Mivechi for the kind gift of packaging cells producing pMV6-hsp-70. [1] Amin, V., Cumming, D.V., Coffin, R.S. and Latchman, D.S., The degree of protection provided to neuronal cells by a pre-conditioning stress correlates with the amount of heat shock protein 70 it induces and not with the similarity of the subsequent stress, Neurosci. Lett., 200 (1995) 85–88. [2] Barbe, M.F., Tytell, M., Gower, D.J. and Welch, W.J., Hyperthermia protects against light damage in the rat retina, Science, 241 (1988) 1817–1820.
[3] Beckmann, R.P., Mizzen, L.A. and Welch, W.J., Interaction of hsp70 with newly synthesized proteins: implications for protein folding and assembly, Science, 248 (1990) 850–854. [4] Chan, P.H., Role of oxidants in ischemic brain damage, Stroke, 27 (1996) 1124–1129. [5] Chen, J., Graham, S.H., Zhu, R.L. and Simon, R.P., Stress proteins and tolerance to focal cerebral ischemia, J. Cerebral Blood Flow Metab., 16 (1996) 566–577. [6] Chopp, M., Chen, H., Ho, K.-L., Dereski, M.O., Brown, E., Hetzel, F.W. and Welch, K.M.A., Transient hyperthermia protects against subsequent forebrain ischemic cell damage in the rat, Neurology, 39 (1989) 1396–1398. [7] Dugan, L.L., Bruno, V.M., Amagasu, S.M. and Giffard, R.G., Glia modulate the response of murine cortical neurons to excitotoxicity: glia exacerbate AMPA neurotoxicity, J. Neurosci., 15 (1995) 4545– 4555. [8] Ferna´ndez-Checa, J.C. and Kaplowitz, N., The use of monochlorobimane to determine hepatic GSH levels and synthesis, Anal. Biochem., 190 (1990) 212–219. [9] Kitagawa, K., Mafsumoto, M., Tagaya, M., Hata, R., Ueda, H., Niinobe, M., Handa, N., Fukunaga, R., Kimura, K., Mikoshiba, K. and Kamada, T., Ischemic tolerance phenomenon found in the brain, Brain Res., 528 (1990) 21–24. [10] Koh, J. and Choi, D.W., Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase efflux assay, J. Neurosci. Methods, 20 (1987) 83–90. [11] Li, G.C., Li, L., Liu, R.Y., Rehman, M. and Lee, W.M.F., Heat shock protein HSP70 protects cells from thermal stress even after deletion of its ATP-binding site, Proc. Natl. Acad. Sci. USA, 89 (1992) 2036– 2040. [12] Liu, Y., Kato, H., Nakate, N. and Kogure, K., Temporal profile of heat shock protein 70 synthesis in ischemic tolerance induced by preconditioning ischemia in rat hippocampus, Neuroscience, 56 (1993) 921–927. [13] Nishimura, R.N., Dwyer, B.E., Clegg, K., Cole, R. and deVellis, J., Comparison of the heat shock response in cultured rat cortical neurons and astrocytes, Mol. Brain Res., 9 (1991) 39–45. [14] Nowak, T.S. Jr., Synthesis of heat shock/stress proteins during cellular injury, Ann. N. Y. Acad. Sci., 679 (1993) 142–156. [15] Papadopoulos, M.C., Sun, X.Y., Cao, J., Mivechi, N.F. and Giffard, R.G., Over-expression of HSP70 protects astrocytes from combined oxygen-glucose deprivation, NeuroReport, 7 (1996) 429–432. [16] Rordorf, G., Koroshetz, W.J. and Bonventre, J.V., Heat shock protects cultured neurons from glutamate toxicity, Neuron, 7 (1991) 1043–1051. [17] Sa´ez, G.T., Bannister, W.H. and Bannister, J.V., Free radicals and thiol compounds: the role of glutathione against free radical toxicity. In J. Vina (Ed.), Glutathione Metabolism – Physiological Functions, CRC Press, Boca Raton, FL, 1990, pp. 237–254. [18] Sharp, F.G. and Sagar, S.M., Alterations in gene expression as an index of neuronal injury: heat shock and the immediate early gene response, Neurotoxicology, 15 (1994) 51–60. [19] Traystman, R.J., Kirsch, J.R. and Koehler, R.C., Oxygen radical mechanisms of brain injury following ischemia and reperfusion, J. Appl. Physiol., 71 (1991) 1185–1195. [20] Williams, R.S., Thomas, J.A., Fina, M., German, Z. and Benjamin, I., Human heat shock protein 70 (hsp70) protects murine cells from injury during metabolic stress, J. Clin. Invest., 92 (1993) 503–508.
HSP70 protects murine astrocytes from glucose deprivation injury Lijun Xu, Rona G. Giffard* Department of Anesthesia, S272, Stanford University School of Medicine, Stanford, CA 94305-5117, USA Received 22 November 1996; revised version received 31 January 1997; accepted 3 February 1997
Abstract Expression of the 70 kDa heat shock protein (HSP70) induced by a first insult is associated with protection from a subsequent ischemic insult in brain. Expression of the human inducible HSP70 was previously shown to protect astrocytes in primary culture from combined oxygen-glucose deprivation. These studies have now been extended to demonstrate that HSP70 expression also protects from isolated glucose deprivation. Slight protection was seen against hydrogen peroxide (H2O2) exposure. Glutathione levels decrease less after glucose deprivation or H2O2 exposure (200 mM) in the cells overexpressing HSP70, compared to either b-galactosidase expressing or uninfected controls (P , 0.01). These data suggest that the HSP70-expressing cells suffered less oxidative stress since their glutathione levels were better preserved. 1997 Elsevier Science Ireland Ltd. Keywords: Retrovirus; Glutathione; Hydrogen peroxide; Mouse; Hypoglycemia; Primary culture; Oxidative stress; Heat shock proteins
Heat shock proteins, and the inducible member of the approximately 70 kDa family, HSP70, in particular, have attracted interest as potential neuroprotectants because prior induction is associated with protection of cultured cells against several different injuries [1,2,11,15,16,20]. Brief pretreatment with ischemia has been shown to protect against both global [9] and focal ischemia [5] and is associated with induction of the heat shock protein HSP70. We have previously reported that expressing human inducible HSP70 using a retroviral vector protects astrocytes in primary culture from combined oxygen-glucose deprivation or a hyperthermic insult [15]. In this paper, we have investigated the range of insults HSP70 expression can protect against by testing glucose deprivation, and hydrogen peroxide (H2O2) exposure. Each represents one aspect of the injury caused by ischemia. While H2O2 is a pure oxidative insult, glucose deprivation represents both an oxidative stress and the loss of substrate. Glutathione (GSH) plays an important role in protecting cells from oxidative stress and in free-radical scavenging [17]. For this reason, we determined the effect of HSP70 overexpression on the decrease in GSH levels caused by these two insults. * Corresponding author. Tel.: +1 415 7258482; fax: +1 415 7258052; e-mail: [email protected]
Primary astrocyte cultures were prepared from postnatal (days 1–3) Swiss Webster mice (Simonsen, Gilroy, CA, USA) as previously described [7]. All procedures were carried out according to a protocol approved by the Stanford University animal care and use committee and were in keeping with the NIH guide. In brief, animals were anesthetized with halothane, decapitated, and the neocortical tissue was freed of meninges and blood vessels, before being minced and suspended in Eagle’s minimal essential medium (MEM; Gibco, Grand Island, NY, USA) supplemented with 21 mM glucose and containing 0.09% trypsin for 20 min at 37°C. The dissociated cells were washed, triturated and plated in 15 mm Falcon Primaria 24-well plates (Becton Dickinson, Lincoln, IL, USA) at a density of 1.8 hemispheres/multiwell (about 2 × 105 cells/cm2) in plating medium consisting of MEM supplemented with 10% equine serum (Hyclone, Logan, UT, USA), 10% fetal bovine serum (Hyclone), 21 mM glucose (Sigma, St. Louis, MO, USA) and 10 ng/ml epidermal growth factor (Sigma). Astrocytes were maintained in a 37°C humidified incubator with a 5% CO2 in room air atmosphere. Immunocytochemical studies revealed that more than 95% of the cells in our cultures stained for glial fibrillary acidic protein. Retroviruses were produced in W-2 packaging cells and
0304-3940/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940 (97 )1 3444-9
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used as previously described [15]. Control retrovirus MPZen-lacZ expressing b-galactosidase from the 5′ LTR promoter and neomycin resistance from an SV40 promoter was the kind gift of Dr. David Vaux. Packaging cells producing pMV6-hsp-70 [11], to express the human inducible HSP70 was the kind gift of Dr. Nahid Mivechi. Cultures were infected 36–48 h after plating by exposure to medium from the packaging cells (after 0.2 mm filtration) and containing 8 mg/ml polybrene (Sigma). Infections were repeated twice. Cells expressing the viral genes were selected in 1 mg/ml G418 for 5 days. All uninfected astrocytes died after 5 days of exposure to G418. The infected astrocytes became essentially confluent within 2 weeks, during which time they were fed twice weekly with plating medium containing 100 mg/ml G418. Cells were used for experiments between days 25–45 in vitro. Expression of HSP70 was confirmed by immunohistochemical staining and immunoblot as previously described [15]. Both cells infected with a retrovirus directing expression of b-galactosidase and uninfected astrocytes, which were not exposed to G418 selection, were used as controls. Experiments were performed at least three times on cultures from a minimum of three different dissections. Astrocyte injury was estimated morphologically by phase-contrast light microscopy and quantitated by measuring lactate dehydrogenase (LDH) activity released from lysed cells into the medium [10]. Total LDH release corresponding to complete astrocyte death was determined at the end of each experiment following freezing at −70°C and rapid thawing. GSH was measured before and after glucose deprivation (GD) for 10 h as described by Ferna´ndez-Checa and Kaplowitz [8] with several modifications. Astrocyte cultures were washed twice with phosphate-buffered saline (PBS), incubated at 37°C for 10 min with 50 mM monochlorobimane (Molecular Probes, Eugene, OR, USA), then washed with PBS and lysed in 0.2% Triton X-100. The insoluble debris was sedimented at 3000 × g for 5 min. The concentration of protein in the supernatant was determined using the bicinchoninic acid method (Pierce, Rockford, IL, USA). The fluorescence of monochlorobimane in the supernatant was measured at 400 nm excitation, 480 nm emission in a Perkin Elmer LS50B spectrofluorimeter. The concentration of GSH was calculated from standard curves and expressed as nmol/mg protein. The relationship between GSH and fluorescence at 480 nm was linear in the range found in the cells. The GSH measurements were also performed after 1 h exposure to 200 mM H2O2, an insult resulting in little cell death. Cultures were deprived of glucose by replacing the culture medium with balanced salt solution (BSS0), at pH 7.4, containing (in mM) NaCl 116, CaCl2 1.8, MgSO4 0.8, KCl 5.4, NaH2PO4 1, NaHCO3 14.7, N-(2-hydroxyethyl)piperazine-N′-(ethanesulfonic acid) (HEPES) 10; and phenol red 10 mg/l lacking glucose. The medium was replaced in each well three times with 800 ml BSS0 resulting in a greater than
2000-fold dilution of growth medium. The medium was sampled for LDH after 10, 18, and 24 h of GD. Astrocyte cultures had their medium exchanged three times with balanced salt solution containing glucose (BSS5.5; BSS0 containing 5.5 mM glucose) at pH 7.4. H2O2 (30%; Sigma) was diluted in the same solution and added to the wells to achieve a final concentration of 200 or 400 mM. The medium was sampled for LDH release after 2, 4, and 6 h of exposure. Values shown are the mean ± SE. Statistical significance (P , 0.05) was determined by ANOVA and Student Neuman–Keuls test for multiple comparisons using SigmaStat (Jandel Scientific). Astrocytes overexpressing HSP70 were significantly more resistant to glucose deprivation than b-galactosidase expressing and uninfected controls, morphologically (Fig. 1) and as assessed by LDH release (Fig. 2A). Astrocytes
Fig. 1. Astrocytes are protected from glucose deprivation (GD) by overexpression of HSP70. Cultures were deprived of glucose by replacing the culture medium with balanced salt solution lacking glucose (BSSo), at pH 7.4. After 24 h, the cells were stained with trypan blue. Pictures were taken at 200 × magnification of the same field with phase contrast (A,C,E) and bright field (B,D,F) optics (bar, 25 mm). HSP70-infected astrocytes were slightly injured and very few cells died (A,B). Cells were almost completely dead and began to peel off in b-galactosidase-expressing cultures (C,D); and uninfected control cultures (E,F).
L. Xu, R.G. Giffard / Neuroscience Letters 224 (1997) 9–12
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overexpressing HSP70 showed no significant protection from 4 or 6 h of 400 mM H2O2 exposure (Fig. 2B), but a small but statistically significant reduction of injury was observed at 2 h. As a measure of the cells’ level of oxidative stress, we then examined the drop in GSH levels after each of the two injury paradigms (Fig. 3). GSH levels were not different among the three groups of astrocytes under normal growth
Fig. 3. GSH was measured before and after 10 h GD or 1 h H2O2 (200 mM) exposure in astrocytes overexpressing HSP70 or b-galactosidase, and uninfected controls. GSH values were standardized to protein. The level of GSH was not different between groups before injury. GSH was significantly decreased in b-galactosidase-expressing and uninfected controls (*P , 0.01), but no significant decrease was evident in the HSP70infected astrocytes after 10 h GD. GSH was significantly higher after GD in HSP70 expressing cultures compared to the b-galactosidase-expressing (*) and uninfected control (#) astrocytes (P , 0.05). All three groups of astrocytes showed significant decreases in GSH after 1 h exposure to H2O2 (P , 0.01) compared to the level in growth medium. However, GSH levels in HSP70 expressing astrocytes were higher than in the other two groups after this insult (P , 0.05, n = 12, mean ± SE).
Fig. 2. (A) Overexpression of HSP70 protects astrocytes from GD. Cultures were washed into balanced salt solution lacking glucose for 24 h. Each point represents the mean of the LDH release (n = 20, mean ± SE) expressed as a percent of maximum LDH release measured after freeze thaw. The HSP70 values are significantly different (P , 0.05) from the bgalactosidase (*) and control (#) values. (B) Effect of HSP70 expression on 400 mM H2O2 injury. Astrocytes which overexpress HSP70, b-galactosidase, and uninfected controls were exposed to 400 mM H2O2 (final concentration in each well) in BSS5.5 for 6 h. LDH release shows that astrocytes are protected by HSP70 expression only at 2 h compared to b-galactosidase-expressing (*) and uninfected control (#) astrocytes (P , 0.05, n = 12, mean ± SE).
conditions. However, after 10 h of glucose deprivation, GSH levels did not significantly decrease in the HSP70 expressing cells while significant drops in both the b-galactosidase (20.1 ± 3.5% decrease) and control astrocytes (30.8 ± 5.4% decrease) were found. GD of 10 h was chosen to measure GSH because at this time the cells were still intact as evidenced by ability to exclude trypan blue, and detection of less than 10% LDH release into the medium. Levels of GSH after exposure to 200 mM H2O2 for 1 h were significantly lower than those in normal growth medium in all three groups. However, the HSP70-expressing cells showed the smallest decrease, 35.6 ± 2.5%, when compared with b-galactosidase expressing cells, 52.8 ± 4.8% decrease, or control uninfected cells, 46.6 ± 3.2% decrease. The difference between groups after H2O2 was significant at P , 0.05. After 1 h exposure to 200 mM H2O2 cell death was less than 10% in HSP70 and bgalactosidase expressing astrocytes, and less than 25% in control astrocytes by both LDH release and trypan blue staining. Both constitutive and inducible members of the HSP70 family of heat shock proteins play a role as molecular chaperones, assisting during protein synthesis and maintaining proteins in a conformation appropriate for transport into subcellular organelles [3,14]. The inducible form of HSP70 is difficult to detect in rat brain in the absence of stressful conditions by immunochemical assay [12,14]. Induction of HSP70 may also be a sensitive marker of
12
L. Xu, R.G. Giffard / Neuroscience Letters 224 (1997) 9–12
acute stress such as ischemia [18]. Heat shock protein induction caused by heat or brief ischemia is associated with protection from a second insult in brain [5,6,12]. As we and others previously found, HSP70 is induced in response to heat and combined oxygen-glucose deprivation in cultured murine cortical astrocytes [13]. In this paper, we found that isolated increases in HSP70 expression were sufficient to provide protection against glucose deprivation and, to a minor extent, H2O2 exposure. Both of these insults reflect subsets of the injury that occurs during cerebral ischemia. Since both of these insults involve oxidative stress, we measured glutathione levels and found that cells protected by overexpression of HSP70 were more resistant to the fall in GSH induced by these injuries. After 10 h glucose deprivation HSP70expressing cells did not show any decrease in GSH, whereas GSH in the b-galactosidase expressing and control astrocytes dropped significantly. Similarly, after H2O2 exposure the HSP70 expressing cells showed the smallest decrease. No difference was evident in levels of GSH in the three groups of astrocytes under normal growth conditions. Our findings demonstrate a negative relationship between the level of GSH and injury. Decreased injury by glucose deprivation or H2O2 exposure was observed in concert with higher GSH levels. One possible explanation of this observation is that overexpression of HSP70 leads to decreased demand for new protein synthesis in response to stress. This can lead to better maintenance of amino acid precursor pools and ATP, both of which are needed for GSH synthesis. Higher levels of GSH synthesis could lead to protection from oxidative insults. During glucose deprivation or H2O2 exposure, cells are subjected to oxidative stress sufficient to be cytotoxic. Reactive oxygen species are also important mediators of hypoxic-ischemic central nervous system injury [4,19]. Our results show HSP70 overexpression in astrocytes is sufficient to protect from glucose deprivation and allows maintenance of higher GSH levels. This work was supported in part by NIH grant GM 49831 to R.G.G. The authors wish to thank Dr. David Vaux for the kind gift of packaging cells producing MPZen-lacZ, and Dr. Nahid Mivechi for the kind gift of packaging cells producing pMV6-hsp-70. [1] Amin, V., Cumming, D.V., Coffin, R.S. and Latchman, D.S., The degree of protection provided to neuronal cells by a pre-conditioning stress correlates with the amount of heat shock protein 70 it induces and not with the similarity of the subsequent stress, Neurosci. Lett., 200 (1995) 85–88. [2] Barbe, M.F., Tytell, M., Gower, D.J. and Welch, W.J., Hyperthermia protects against light damage in the rat retina, Science, 241 (1988) 1817–1820.
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