HSP70 is essential to the neuroprotective effect of heat-shock

BRAIN RESEARCH Brain Research 740 (1996) 117-123

ELSEVIER

Research report HSP70 is essential to the neuroprotective effect of heat-shock K a o r u Sato, H i r o s h i Saito, N o r i o M a t s u k i ~' Department 0t('hemico/Plmrmacology. Faculty of'Pharmaceutical Sciences, The Uniuer.~iO"ql'Tokvo. 7-3-1 Hon~:o. Bunkvo-ku. 7)~kvo 113. ,lapan Accepted 2 July 1996

Abstract Many kinds of injuries induce 72 kDa heat-shock protein (HSP70) in the central nervous system. We investigated the role of HSP70 in promoting the survival of rat hippocampal neurons in primary culture. Heat-shock (42°C for 30 min) significantly increased the number of surviving neurons independently of the initial density of plated cells, suggesting a direct effect on the neurons, hnmunohistochemical detection revealed that HSPT0 was expressed in virtually all cells six hours after the heat-shock and the immunostaining became stronger during the observation period of 72 h. HSP70 immunoreactivity was localized in the nucleus at 24 h after the heat-shock, but was diffused throughout the cytoplasm at 72 h. Addition of an antisense oligonucleotide to the medium significantly suppressed the neuroprotective effect of the heat-shock to control level, while a sense oligonucleotide had no effect. HSPT0 immunoreactivity was completely abolished in the presence of the antisense oligonucleotide. These results indicate that HSP70 is essential for neuroprotection by heat-shock. Kuywords: Heat-shock: Stress protein; Survival; Neuroprotection; Antisense oligonucleotide: Cultured neuron

1. Introduction

Cells synthesize a group of proteins called heat-shock proteins (HSP) when exposed to various kinds of stress, such as high temperature, low temperature, heavy metals and radiation, and this response has been well conserved throughout evolution. 72-kDa heat-shock protein (HSP70), a member of the HSP70 family, which is the most abundant and best conserved subset of eukaryotic stress proteins [30], is synthesized transiently following stress and is thought to play some functional role as a molecular chaperon. In the central nervous system (CNS), many kinds of injuries, such as ischemia [16,24,40,54], trauma [11,50], seizures [17,52,53] and Alzheimer's disease [18], induce HSP70 in vivo. However, the pattern of HSP70 expression does not always parallel the temporal and regional pattern of cell survival, and the physiological significance of the response for brain tissue is not clear. Several studies have shown that priming with moderate stress resulted in an attenuation of neuronal injury after subsequent insult [7,12,25,26,38,57]. In vitro studies also indicated that brief hyperthermic stress could induce tolerance to glutamate toxicity [32,44]. These phenomena were associated with induction of the synthesis of HSP70 by the prior stress, and the implication of these studies was that the induced

* Corresponding author, Fax: + 81 (3) 3815 4603.

HSP70 contributed to the neuroprotective action. No direct evidence of this is available. In recent years, antisense oligonucleotide techniques have been developed as powerful experimental tools to block protein synthesis with high specificity. Although the mechanisms by which gene expression is inhibited are poorly understood, several reports have shown that synthetic antisense oligonucleotides downregulate translation in vitro [22,59]. In the study presented here, we characterized the effect of heat-shock on the viability of hippocampal cells cultured in serum-free medium, and investigated the role of HSP70 directly by blocking its synthesis with a specific antisense oligonucleotide. We also examined the expression of HSPT0 by an immunocytochemical procedure to confirm both its induction by heat-shock and the effect of the antisense oligonucleotide.

2. Materials and methods 2. I. Cell culture

Dissociated brain neurons were cultured as described elsewhere [3,35]. Briefly, whole brains were isolated from embryonic day 18 Wistar rats, and the hippocampal regions were dissected out. Each tissue was treated with 0.25% trypsin and 0.01% DNase I at 37°C for 20 rain.

(t006-8993/96/$15.00 Copyright :(: 1996 Elsevier Science B.V. All rights reserved. I'll SI)(11)6 ,~t)~t~I96)l)0846 3

118

K. Sato et al. / Brain Rescar~'h 740 ( /990J 1 / 7 /2:;

After termination of the incubation by the addition of heat-inactivated horse serum, the tissue fragments were centrifuged at 1200 rpm for 5 rain. The pellet was resuspended in modified Eagle's medium supplemented with 10% fetal bovine serum, and cells were dissociated by gently passing the suspension through a plastic-tipped pipet. The cell suspension was diluted, and plated on 48-well plastic plates (1 crn-~/well) coated with poly-Llysine to give a density of 1 × 105 cells/cm 2 or 5 × 10 ~ cells/cm 2, and cultured at 37°C in a humidified 5% CO~-95% air atmosphere. At 24 h after plating, cells were heat-shocked at 42°C for 30 rain, then the medium was changed to serum-free DF medium (1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 supplemented with N2 hormones) and incubation was continued at 37°C. Three days after the change of medium, the cell cultures were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (PB). The neuronal cells were visualized by Nissl staining with Cresyl violet or by specific immunostaining with monoclonal antibody to microtubule-associated protein 2 (MAP2). Ten areas of 1 mm 2 were chosen at random from each well, and the surviving neurons were counted under a microscope. The number of neurons was finally expressed as cells/cm 2. Sense or antisense oligonucleotide was added to the serum-free DF medium at the concentration of 0.1 or 1 ~ M immediately after heat-shock when the medium was changed.

..6o [ f Z 4° )

"

*-

I

~ 20 ~-

°

0/ Cont Heat CS-23 **:p
Fig. 1. Effect of heat-shock on hippocampal neurons plated at I × I0 > c e l l s / c m e. Cells were exposed to a heat-shock of 42°C for 30 nlin followed by a change of the medium and incubation for 72 h at 37°C. Similar restllts were obtained in two uther scts of experimcnts (data not shown). Each column and bar represents the m e a n ± S . E . M , fi'om four wells. ' ' P < 0.01 vs. control (Dunnet's test).

2.3. Sense and antisense oligonucleotides for HSP70 A specific antisense oligonucleotide was synthesized based on a highly conserved region [37] of the recently sequenced rat hsp70 cDNA [31] corresponding to nucleotides 550-565 of the sequence, as reported by

2.2. hnmunocytochemist~ A mouse monoclonal antibody against HSP70 (C92F3A-5) was obtained from Stress Gen Biotechnologies Corp. (Victoria, BC, Canada). This monoclonal antibody specifically recognizes stress-inducible HSP70 [32,48,54,58]. Cultured cells were rinsed gently three times with 10 mM phosphate-buffered 0.15 M NaC1, pH 7.4 (PBS) for 5 min each, and fixed with 4% paraformaldehyde in PB containing 4.5% sucrose for 30 min. The dishes were then washed with PBS three times for 10 min each, with 0.3% Triton X-100 in PBS for 30 min and then with PBS three times, followed by overnight blocking with 10% normal horse serum in PBS at 4°C. The cells were exposed for 8 h to the monoclonal antibody C92F3A-5 diluted 1:800 in PBS containing 0.5% BSA at 4°C, followed by an overnight wash in PBS. Biotinylated horse anti-mouse IgG (Vectastain Elite ABC kit. Vector, Burlingame, CA), was added for 60 min, then the cells were washed with PBS three times for 10 rain each. Endogenous peroxidase was blocked with 0.3% H202 in MeOH for 60 min then the cells were again washed thoroughly. They were incubated with avidin-biotin-peroxidase complex (Vectastain Elite ABC kit, Vector, Burlingame, CA) for 60 min, washed with PBS three times for 5 min each, and exposed to a solution of 0.2 m g / m l diaminobenzidine (Dojindo, Japan) and 0.5% H202 in PB.

Fig. 2. Typical photomicrographs of hippocampal cells cuhured tier 72 h m serum-free DF medium under control conditions (A) or heal-shock conditions (B). The horizontal bar indicates 100 ,am.

119

K. Sato et al. / Brain Research 740 (1996~ 117 123 3O

A. n e u r o n 2o O

x

.z lO 09

o

0 Cont

Heat Shock

~ 30

B. glia 20

.

Fig, 5. Typical photomicrograph showing nuclear localization of HSPTO 24 h after the heat-shock. The horizontal bar represents 50 ,am.

~0

o

0 Cont

Heat Shock

nucleotides were 5'-CTTGGTCAGCACCATG-3'

"p<0 05, **:p<0 01 vs Control (Student's t test)

Fig. 3. Effect ot heat-shock on hippocampal cells plated at low density (5 X 10 :~ cells/era ~ ). The numbers of surviving neurons and glial cells were counted separately. The number of wells was flmr each. * P < 0.05, • ' P < 0.01 vs. control (Student's t-test).

CATGGTGCTGACCAAG-3'.

a n d 5'-

respectively.

3. R e s u l t s

K a t a y a m a et al. [22]. C o n s t i t u t i v e l y e x p r e s s e d rat h s p - r e l a t e d p r o t e i n ( H S C 7 0 ) is m i s m a t c h e d b y f o u r o l i g o n u c l e o tides. T h e

sequences

of the antisense and

sense oligo-

A s s h o w n in Fig. 1, a h e a t - s h o c k o f 4 2 ° C for 30 rain clearly

promoted

the

survival

of hippocampal

neurons

g

*

Fig. 4. HSPTI) immunoreactivity 72 h after the heat-shock and the effect of sense and antisense oligonucleotides to that. A: control ceils withom heat-shock: B: heat-shocked cells: C: heat-shocked ceils treated with sense oligonucleotide: D: heat-shocked cells treated with antisense oligonucleotide. Photographs were taken at 72 h after changing the medium. The horizontal bar represents I00 p,m.

K. Sam et al. / Brain Re.search 740 (197~) /17 123

120

cultured at the density of 1 × 105 cells/cm 2. In the control, only 24% of the plated neurons survived for 72 h. The cause of neuronal death in serum-free condition may be a loss of trophic factors). As a positive control, we employed CS-23, or recombinant basic fibroblast growth factor (bFGF), in the DF medium at the concentration of 10 ng/ml, which strongly enhances the neuronal survival [3]. The protective effect of heat-shock was very strong and comparable to that of CS-23. Typical photomicrographs of surviving cells at 72 h after heat-shock are shown in Fig. 2. Many glial cells surround the neurons. Since glial cells are known to afford protection to neurons in many injurious situations [36,45,56], it seemed possible that the protective effect of heat-shock was not due to a direct action on neurons, but rather was an indirect effect mediated by glial cells. Therefore, we decreased the initial cell density to 5 × 103 cells/cm 2 (Fig. 3). At this density, individual cells are isolated, and the number of glial cells could be counted. Heat-shock-induced neuronal protection was retained at this density (Fig. 3A), and the number of glial cells was also increased (Fig. 3B). These data suggest that heat-shock directly affects both neurons and glias, when it enhances their survival. We investigated immunohistochemically whether the heat-shock induced HSP70 (Fig. 4A,B). A low level of HSP70 was detected in almost all cells 6 h after the heat-shock (data not shown) and it became intense at 72 h (Fig. 4B). Although we did not make a systematic examination, HSP70 immunoreactivity appeared to be present in both neurons and glial cells. In the control, HSP70 was not detectable up to 72 h after changing the medium. Interestingly, at 24 h after the heat-shock, prominent nuclear localization of HSP70 was observed (Fig. 5), but the

100

&--, E 8o

~ ~o ~ 40

g~ N

o 20

o DF

0.1

1 Sense

[ - - - - I Control Heat shock

0.1 1 Antisense

(oM)

** : p<0.01 (Duncan's test)

Fig. 6. Effects of oligonucleotides on enhancement of survival by heatshock. Sense and antisense oligonucleotides were added to serum-free DF medium just after the heat-shock. Open and cross-hatched columns denote control and heat-shocked neurons, respectively. The second column from the left (DF) indicates the results ot" changing the medium without oligonucleotide. The number of wells was four each. * P < 0.05, • P < 0.01 vs. control (Duncan's test).

staining was distributed over the whole cell al 72 h (Fig. 4B). To obtain direct evidence that induction of HSP70 is essential to the heat-shock-induced protective effect, an antisense oligonucleotide was applied to the serum-free I)F medium ,just alter the heat-shock (Fig. 6). In a preliminary experiment, we examined the effect of antisense and sense oligonucleotides under control conditions. When the concentration was higher than 100 /,tM, both oligonucleotides were apparently toxic. Therefore, we employed the concentrations of 0.1 and I /*M. Under control conditions, the sense and antisense oligonucleotides had no effect on the neuronal survival. Under heat-shock conditions, lhe antisense, but not the sense, oligonucleotide suppressed the neuroprotective effect of heat-shock to the control level. We confirmed immunohistochemically that antisense oligonucleotide actually blocked the synthesis of HSP70 (Fig. 4). Application of either oligonucleotide had no effect on the control immunoreactivity (data not shown). Heat-shock-induced HSP70 synthesis (Fig. 4B) was completely abolished in the presence of the antisense oligonucleotide (Fig. 4D). while the sense oligonucleotide had no effect (Fig. 4C). 4. Discussion

Heat-shock of 42°C for 30 min markedly enhanced the survival of cultured hippocampal neurons. The protective effect of heat-shock was retained at low cell density (5 X 103 cells/cm2), and the number of glial cells was also increased. These data suggest that heat-shock directly affected both neurons and glias. We have been investigating drugs and endogenous substances that promote the survival of primary cultured neurons, and have shown that bFGF [3], sperrnine [1,13], interleukin-2 [47], epidermal growth factor [2,4], and a-tocopherol [48] are neuroprotective. Among them bFGF showed the strongest effect. Interestingly, the survival-promoting effect of the heatshock was comparable to that of bFGF, though the effect of bFGF was stronger on glial cells, while that of the heat-shock was apparently equal on neurons and glia. We confirmed immunohistochemically that HSP70 was induced when the heat-shock enhanced the survival of hippocampal cells. The immunoreactivity was observed in both neurons and glial cells at the same level. This is the first time that expression of HSP70 in cultured hippocampal cells has been demonstrated. Lowenstein et al. [32] reported the induction of HSP70 in cerebellar granule neurons and glial cells by heat-shock. However, Marini et al. [34] were unable to detect HSP70 expression in neurons, despite its expression in astrocytes under similar culture conditions after a slightly different heat-shock treatment. These results suggest that there may be a stress threshold for HSP70 induction and it may differ in different cellular populations. In oligodendrocytes and astrocytes HSP70 induction was heterogeneous with respect to the

K. Sato et al. / Brain Research 740(1996) 117-123

quantity and time course [49]. This report supports the above idea. Of course, transcriptional or post-transcriptional mechanisms involved in HSP70 induction may vary among cell types, but recent studies have demonstrated the implication of HSP70s in the regulation of the heat-shock response, i.e. the basal amount of HSC70 is one of the factors that modulate the level of HSP70 expression [5,8,14,15,21,39]. A high level of HSC70 m R N A in neuronal cell populations and a much lower level in glial cells have been reported [42]. Based on the above findings, there seem to be two possible explanations for our observations. First, in hippocampus all cell populations may have the same stress threshold. Second, although the different cell populations have different thresholds, the heatshock conditions of this experiment were severe enough to cause all the cells to induce HSP70. More experiments under various heat-shock conditions should be carried out, and measurement of the basal level of HSC70 is also necessary. The intracellular distribution of HSP70 changed with time. At 24 h after heat-shock, HSP70 was concentrated strongly in the nucleus (Fig. 5), while at 72 h, it was diffused throughout the cell (Fig. 4B). Although such nuclear translocation is well established [55], the molecular and biochemical basis underlying this phenomenon and its functional significance are unknown. The removal of HSP70 C-terminal amino acids, which are necessary for localization, abrogated the protective effect, whereas the removal of N-terminal amino acids, which are necessary for ATP binding, caused no change [28]. Using immunoelectron microscopy and a sensitive silver staining technique, Welch and Suhan [58] showed that nuclear HSPT0 mostly resides within the granular region of the nucleolus and they proposed an interaction with ribosomal particles. To investigate more directly the role of HSPT0 in the heat-shock-induced enhancement of neuronal survival, we employed an antisense oligonucleotide that specifically blocked the synthesis of HSP70. The antisense oligonucleotide significantly suppressed both heat-shock-induced expression of HSP70 (Fig. 4D) and the enhancement of neuronal survival (Fig. 6). Based on an estimate of approximately 3 - 4 billion base pairs in the human genome and assuming a random distribution of bases, the minimum size of an antisense oligonucleotide needed to recognize a single specific sequence in the genome is between 12 and 15 bases [19], We synthesized a 16-mer antisense oligonucleotide based on a region that is highly conserved between human and Drosophila [37]. It mismatches the sequence of HSC70 by four nucleotides and Miller et al. [37] showed that the same oligonucleotide probe did not cross-hybridize with the HSC70 mRNA. Katayama et al. [22] indicated by immunoblotting that this antisense oligonucleotide blocked the synthesis of HSP70 in neuroblastoma × glioma hybrid cells. Therefore, our antisense oligonucleotide is considered to suppress the expression of HSP70 specifically.

121

Several efforts have been made to identify whether HSP70 is protective or not in vitro. Microinjection of antibodies specific to HSP70 into rat fibroblasts rendered these cells thermosensitive [43]. Microinjection of HSPT0 m R N A into mouse oocytes increased their thermotolerance [20]. As regards neuroprotection, microinjection of antibodies reduced the survival of cultured mouse neurons after severe heat-shock [23]. In addition, transfection of rat dorsal root ganglia with human HSP gene enhanced their thermotolerance [51]. Our data are consistent with the published data and provide the most direct evidence to date that HSP70 has a protective role in neurons. Previous reports showed that the prior heat-shock partially protected neuroblastoma × dorsal root ganglion hybrid cells from apoptotic cell death alter transfer to serum-Dee DF medium [33]. However, no morphological characteristic of apoptosis, such as membrane blebbing or nuclear condensation tk~llowed by explosive fragmentation, was observed in our experiment. Therefore, in our experiment, HSPT0 probably contributes to the protection of neural cells from necrosis. HSP70 is known to associate with newly synthesized, unfolded or aberrantly folded proteins as a molecular chaperon [9,10,41]. It might act to stabilize denatured proteins and to prevent inappropriate protein interactions in the course of necrosis. HSPT0 also has the ability to bind to cytoskeletal proteins [6,27,29,46], so it is possible that HSP70 stabilized the neuronal cytoskeleton and protected it from degradation. Control of the level of HSP70 might be a new strategy for brain protection in the clinical context. In conclusion, the present results indicate that HSP70 plays an essential role in the neuroprotective cffec! of heat-shock. Its action is very strong.

References [1] Abe, K., Chida, N., Nishiyama, N. and Saito, H., Spermine pro rnotes the survival of primary cultured bruin neurons, Brain Res., 605 (1993) 322 326. [2] Abe, K., Takayanagi, M. and Saito, H., A comparison of neu rotrophic effects of epidermal growth factor and basic fibroblust growth factor in primary cultured neurons I'ronl vurious regions of fetal rat brain, .Ipn. J. Pharm~lcol., 54 (1990) 45-51. [3] Abe, K.. Takayanagi, M. and Saito, H., Effect of recombinant humun basic fibmblast growth factor and its modified protein CS23 on survival of primary cultured neurons from various regions of fetal rat brain, Jpn. ,1. Pharmacol., 53 (1990) 221- 227. [4] Abe, K.. Takayanagi, M. and Saito, H.. Neurotrophic effects of epidermal growth factor on cultured brain neurons are blocked by protein kinase inhibitors, Jpn, .1. Pharmacol., 59 (1992) 259-261. [5] Abravaya, K., Myers, M.P., Murphy, SR and Morimoto, R.I., The human heat shock protein hsp70 interacts with HSF, the transcrip tion factor that regulates heat shock gene expression. (;,,m,.~ Dec., ~ (1992} ll53 1164. [6] Ahmad, S,, Ahuja, R., Venner. T.J. and Gupta, R.S., [demification of a protein altered in mutants resistant to microtubule inhihilors as u member of the major heal shock protein (hsp70) family, Mol. ('dl. Biol., 10(1990) 5160 5165,

122

K. Sato et al. / Brain Research 740 (1996) 1 l 7-123

[7] 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. [8] Baler, R., Welch, W.J. and Voelmy, R., Heat shock gene regulation by nascent polypeptides and denatured proteins: hsp70 as a potential autoregulatory factor, J. Cell Biol., 117 (1992)1151-1159. [9] Beckrnann, R.P., Lovett, M. and Welch, W.J., Examining the function and regulation of hsp70 in cells subjected to metabolic stress, J. Cell Biol., 117 (1992) 1137-1150. [10] Beckmann, R.P., Mizzen, L.A. and Welch, W.J., Interaction of hspT0 with newly synthesized proteins: Implications for protein folding and assembly, Science, 248 (1990) 850-854. [11] Brown, I.R., Rush, S.J. and Ivy, G.O., Induction of a heat shock gene at the site of tissue injury in the rat brain, Neuron, 2 (1989) 1559-1564. [12] Chopp, M., Cben, H., Ho, K.L., Dereski, M.O., Brown, E., Hetzel, F.W. and Welch, K.M.A., Transient hyperthermia protects against subsequent forebraln ischemic cell damage in the rat, Neurology, 39 (1989) 1396-1398. [13] Chu, P.J., Saito, H. and Abe, K., Polyamines promote neurite elongation of cultured rat hippocampal neurons, Neurosci. Res, 19 (1994) 155-160. [14] Craig, E.A. and Gross, C.A., Is hsp70 the cellular thermometer? TIBS, 16 (1991) 135-140. [15] DiDomenico, B.J., Bugaisky, G.E. and Lindquist, S., The heat shock response is self-regulated at both the transcriptional and posttranscriptional levels, Cell, 31 (1982) 593-603. [16] Ferriero, D.M., Soberano, H.Q., Simon, R.P. and Sharp, F.R., Hypoxia-ischemia induces heat shock protein-like (HSP70) immunoreactivity in neonatal rat brain, Dev. Brain Res., 53 (1990) 145-150. [17] Gonzalez, M.F., Shiraishi, K., Hisanaga, K., Sagar, S.M., Mandabach, M. and Sharp, F.R., Heat shock proteins as markers of neural injury, Mol. Brain Res.. 6 (1989) 93-100. [18] Hamos, J.E., Oblas, B., Pulaski-Salo, D., Welch, W.J., Bole, D.G. and Drachman, D.A., Expression of heat shock proteins in Alzheimer's disease, Neurology, 41 (1991) 345-350. [19] Helene, C. and Thuong, N.T., Control of gene expression by oligonucleotides covalently linked to intercalating agents, Genome, 31 (1989) 413-421. [20] Hendrey, J. and Kola, I., Thermolability of mouse oocytes is due to the lack of expression and/or inducibility of Hsp70, Mol. Reprod. Dev., 28 (1991) 1-8. [21] Hightower, E,L. and Li, T., Structure and function of the mammalian HSP70 family. In J. Mayer and I. Brown (Eds.), Heat Shock Proteins in the Nervous System, Academic Press, San Diego, 1994, pp. 1-30. [22] Katayama, S., Shuntoh, H., Matsuyama, S. and Tanaka, C., Effect of heat shock on intracellular calcium mobilization in neuroblastoma × glioma hybrid cells, J. Neurochem., 62 (1994) 2292-2299. [23] Khan, N.A. and Sotelo, J., Heat shock stress is deleterious to CNS cultured neurons microinjected with anti-HSP70 antibodies, Biol. Cell., 65 (1989) 199-202. [24] Kiessling, M., Dienel, G.A., Jacewicz, M, and Pulsinell, W.A., Protein synthesis in post-ischaemic rat brain: a two-dimensional electropboretic analysis, J. Cereb. Blood Flow Metab., 6 (1986) 642-649. [25] Kirino, R., Tsujita, Y. and Tamura, A., Induced tolerance to ischemia in gerbil hippocarnpal neurons, J. Cereb. Blood Flow Metub., 11 (1991) 299-307. [26] Kitagawa, K., Matsumoto, M., Tagaya, M., Kuwabara, M., Hata, R., Handa, N., Fukunaga, R., Kimura, K. and Kamada, T., Hyperthermia-induced neuronal protection against ischemic injury in gerbils, J. Cereb. Blood Flow Metub., 11 (1991) 449-452. [27] Lewis, V.A., Hynes, G.M., Zheng, D., Saibil, H. and Willison, K., T-complex polypeptide-1 is a subunit of a heteromeric particle in the eukaryotic cytosol, Nature, 358 (1992) 249-252. [28] Li, G.C., Li, L., Liu, R.Y., Rehman, M. and Lee, W.M.F., Heat

[29]

[30] [31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39] [40] [41]

[42]

[43]

[44]

[45]

[46]

[47]

[48]

[49]

shock protein hsp70 protects cells from thermal stress even after deletion of its ATP-binding domain, Proc. Natl. Acad. Sci. USA, 89 (1992) 2036-2040. Liao, J., Lowthert, L.A., Ghori, N. and Omary, M.B., The 70-kDa heat shock proteins associate with glandular intermediate filaments in an ATP-dependent manner, J. Biol. Chem., 270 (1995) 915-922. Lindquist, S,, The heat-shock response, Annu. Rec. Biochem., 55 (1986) 1151-1191. Longo, F.M., Wang, S., Narasimhan, P., Zhang, J.S., Chen, J., Massa, S.M. and Sharp, F.R., cDNA cloning and expression of stress-inducible rat hsp70 in normal and injured rat brain, J. Neurosci. Res., 36 (1993)325-335. Lowenstein, D.H.. Chan, P.H. and Miles, M.F., The stress protein response in cultured neurons: characterization and evidence for a protective role in excitotoxicity, Neuron, 7 (1991) 1053-1060. Mailhos, C., Howard, M.K. and Latchman, D.S., Heat shock protects neuronal cells from programmed cell death by apoptosis, Neuroscience, 55 (1993) 62 1-627. Marini, A.M., Kozuka, M., Lipsky, R.H. and Nowak, T.S., 70-kilodalton heat shock protein induction in cerebellar astrocytes and cerebellar granule cells in vitro: comparison with immunocytochemical localization after hyperthermia in vivo, .L Neurochem., 54 (1990) 1509-1516. Matsuda, S., Saito, H. and Nishiyama, N., Effect of basic fibroblast growth factor on neurons cultured from various regions of postnatal rat brain, Brain Res., 520 (1990) 310-316. Mattson, M.P. and Rycblik, B., Glia protect hippocampal neurons against excitatory amino acid-induced degeneration: Involvement of fibroblast growth factor, Int. J. Dev. Neurosci., 8 (1990) 399-415. Miller, E.K., Raese, J.D. and Morrison-Bogorad, M., Expression of heat shock protein 70 and heat shock cognate 70 messenger RNAs in rat cortex and cerebellum after heat shock or amphetamine treatment, J. Neurochem., 56 (1991) 2060-2071. Mizzen, L.A. and Welch, W.J., Characterization of the thermotolerant cell I. Effects on protein synthesis activity and the regulation of heat-shock protein 70 expression, J. Cell Biol., 106 (1988) 1105 1116. Morimoto, R.I., Cells in stress: Transcriptional activation of heat shock genes, Science, 259 (1993) 1409-1410. Nowak, T.S., Synthesis of a stress protein following transient ischemia in the gerbil, J. Neurochem., 45 (1985) 1635-1641. Palleros, D.R., Welch, W.J. and Fink, A.L., interaction of hspT0 with unfolded proteins: effects of temperature and nucleotides on the kinetics of binding, Proc. Natl. Acad. Sci. USA, 88 (1991) 57195723. Pardue, S., Groshan, K., Raese, J.D. and Morrison-Bogorad, M., Hsp70 mRNA induction is reduced in neurons of aged rat hippocampus after thermal stress, Neurobiol. Aging, 13 (1992) 661-672. Riabowol, K.T., Mizzen, L.A. and Welch, W.J., Heat shock is lethal to fibroblasts microinjected with antibodies against hsp70, Science, 242 (1988) 433-436. Rordoff, G., Koroshetz, W.J. and Bonventre. J.V., Heat shock protects cultured neurons from glutamate toxicity, Neuron, 7 (t 991 ) 1043-1051. Rosenberg, P.A. and Aizenman, E., Hundred-fold increase in neuronal vulnerability to glutamate toxicity in astrocyte-poor cultures of rat cerebral cortex, Neurosci. Lett., 103 (1989) 162-168. Sanchez, C., Padilla, R., Paciucci, R., Zabala, J.C. and Avila, J., Binding of heat-shock protein 70 (hsp70) to tubulin, Arch. Biochem. Biophys., 310 (1994) 428-432. Sarder, M., Saito, H. and Abe, K., Interleukin-2 promotes survival and neurite extension of cultured neurons from fetal rat brain, Brain Res., 625 (1993) 347-350. Sato, K., Saito, H. and Katsuki, H., Synergism of tocopherol and ascorbate on the survival of cultured brain neurones, NeuroReport, 4 (1993) 1179-1182. Satoh, J., Yamamura, T., Kunishita, T. and Tabira, T., Heteroge-

K. Sato et al. / Brain Research 740 (1996) 117--123

[50]

[51]

[521

[53]

!54]

neous induction of 72-kDa heat shock protein (HSP72) in cultured mouse oligodendrocytes and astrocytes, Brain Res., 573 (1992) 37-43. Tanno, H., Nockels, R.P., Pitts, L.H. and Noble, L.J., lmmunolocalization of heat shock protein after fluid percussive brain injury and relationship to breakdown of the blood-brain barrier, J. Cereb. Blood Flow Metab., 13 (1993) 116-124. Uney, J.B., Kew, J.N.C., Staley, K., Tyers, P. and Sofroniew, M.V., Transfection-mediated expression of human Hsp70i protects rat dorsal root ganglion neurons and glia from severe heat stress, FEBS Lett., 334 (1993) 313-316. Uney, J.B., Leigh, P.N., Marsden, C.D., Lees, A. and Anderton, B.H., Stereotaxic injection of kainic acid into the striatum of rat induces synthesis of mRNA for heat shock protein 70, FEBS Lett., 235 (1988) 215-218. Vass, K., Berger, M.L., Nowak, T.S., Welch, WJ. and Lassman, H., Induction of stress protein hsp70 in nerve cells after status epilepticus in the rat. Neurosci. Lett., 100 (1989) 259-264. Vass, K.. Welch. W.J. and Nowak, T.S., Localization of 70-kDa

[55]

[56]

[57]

[58]

[59]

123

stress protein induction in gerbil brain after ischemia., Aeta Neuropathol., 77 (1988) 128-135. Velazquez, J.M. and Lindquist, S., hsp70: Nuclear concentration during environmental stress and cytoplasmic storage during recovery, Cell, 36 (1984) 655-662. Vibulsreth, S., Hefti, F., Ginsberg, M.S.. Dietrich, W.D. and Busto, R., Astrocytes protect cultured neurons tYom degeneration induced by anoxia, Brain Res., (1987) 422 303 311. Welch, W.J. and Mizzen, L.A., Characterization of the thermotolerant cell. 11. Effects of the intracellular distribution of heat-shock protein 70, intermediate filaments, and small nuclear ribonucleoprotein complexes, J. Cell Biol., 106 (1988) 1117-1130. Welch. W.J. and Suban, J.P., Cellular and biochemical events in mammalian cells during and after recovery tYom physiological stress, J. Cell Biol.. 103 (1986) 2035-2052. Zamecnik, P,C. and Stephenson, M.L.+ Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide, Proc. Natl. Acad, Sci, USA, 75 (1978) 280-284.