The stress protein response in cultured neurons: Characterization and evidence for a protective role in excitotoxicity

Neuron,

Vol. 7, 1053-1060, December,

1991, Copyright

0 1991 by Cell Press

The Stress Protein Response in Cultured Neurons: Characterization and Evidence for a Protective Role in Excitotoxicity Daniel H. Lowenstein,*+ and Michael F. Miles*5

Pak H. Ghan,**

*Department of Neurology ‘Epilepsy Research Laboratory *Department of Neurosurgery University of California, San Francisco San Francisco, California 94143 §Ernest Gallo Clinic and Research Center San Francisco General Hospital San Francisco, California 94110

Summary We used purified cultures of cerebellar granule cells to investigate the possible protective role of stress proteins in an in vitro model of excitotoxicity. Initial experiments used one- and two-dimensional polyacrylamide gel electrophoresis to confirm the induction of typical stress protein size classes by heat shock, sodium arsenite, and the calcium ionophore A23187. tmmunoblot analysis and immunocytochemistry verified the expression of the highly inducible 72 kd heat shock protein (HSP72). Granule cell cultures exposed to glutamate showed evidence of cellular injury that was prevented by the noncompetitive NMDA antagonist MK-801, yet glutamate did not induce a detectable stress protein response. Nonetheless, preinduction of heat shock proteins was associated with protection from toxic concentrations of glutamate. These results imply that the HSP72 expression observed in in vivo models of excitotoxicity may not be directly related to the effects of excitatory amino acids. However, the ability of stress protein induction to protect against injury from glutamate may offer a novel approach toward ameliorating damage from excitotoxins. Introduction Recent results from a number of laboratories have suggested that the stress protein response and heat shock proteins (HSPs) may have an important role in the protection of cells from a variety of toxic conditions. While stress proteins are known to be induced by various agents in many different organisms and cell culture systems, limited work has focused on the role of these proteins in the mammalian CNS. Brown and coworkers (Brown, 1983; Freedman et al., 1981), using two-dimensional polyacrylamide gel electrophoresis (PAGE) of in vitro translation products, demonstrated that HSPs were induced in the rabbit CNS by hyperthermia. Similar approaches were used to show the expression of HSPs in the rat brain during postischemic recirculation (Dienel et al., 1986; Jacewicz et al., 1986; Kiessling et al., 1986; Nowak, 1985). Following the development by Welch and Suhan (1986) of a specific monoclonal antibody directed against HSP72, the highly inducible member of the 70

kd family of stress proteins, a number of in vivo studies documented the induction of HSP72 at the cellular level in rodent brain following focal and global ischemia (Vass et al., 1988; Gonzalez et al., 1989, 1991; Dwyer et al., 1989; Ferriero et al., 1990; Simon et al., 1991), trauma (Cower et al., 1989), and seizures (Vass et al., 1989; Lowenstein et al., 1990). These studies have shown the HSP72 is induced in neurons in the areas most affected bythe injury, although with some forms of injury HSP72 is expressed in astrocytes as well (Gonzalez et al., 1991). Since there is considerable evidence that excitatory amino acids have a causal role in the neuronal cell death following ischemia, trauma, seizures, and other forms of CNS injury (Choi, 1988), it has been proposed that the HSP expression observed with these events represents an adaptive molecular responsetoexcitotoxicinjury(Brown,l990).Thequestion remains, however, whether HSP induction is merely a marker for neuronal injury or a form of cellular protection. Studies have suggested that HSP induction during CNS injury may be protective, much like the effects of stress proteins on heat-induced injury. Barbe et al. (1988) provided the first indirect evidence for this protective role of HSPs by showing that preinduction of HSPs in the rabbit retina via mild systemic hyperthermia was associated with a decrease in lightinduced retinal injury. More recent work has shown that mild global ischemiawhich is sufficient to induce HSP72 in the hippocampus will lessen the hippocampal injury associated with a subsequent s’evere ischemit insult (Kirino et al., 1991; Kitagawa et al., 1990). Thus, there is a suggestion that HSP induction can be protective against presumed excitotoxic injury in the CNS. Unfortunately, such in vivo studies allow for many other factors to complicate the results. For example, direct effects of hyperthermia or prior ischemia on the vascular supply might provide a protective effect that is unrelated to HSP induction within neurons. To explore the function of stress proteins in CNS injury, we have characterized the stress protein response of highly purified cultures of cerebellar granule cells. The experiments presented here were designed to address three specific questions. First, how does the repertoire of stress proteins in neurons compare with that described in nonneuronal cells? Second,doesexcitotoxicity in vitro induce HSP72orother stress proteins in neuronal cultures, as occurs with in vivo models of injury? Finally, does stress protein induction offer protection against excitotoxic injury in an in vitro system? Our results show that cultured neurons have the capacity to express HSP72 and a variety of other stress proteins in response todifferent stimuli. Surprisingly, exposure of neurons to the excitotoxin glutamate did not induce a detectable stress protein response.

Neuron 1054

Nonetheless, induction of HSPs was associated with protection from glutamate toxicity. These results demonstrate that neurons possess a functional stress protein response and expand the potential approaches for reversing excitotoxicity.

A

.

Heat shock /

Arsenlte 1, \

Results Stress Proteins Are induced in Neurons by Heat Shock, Sodium Arsenite, and a Calcium lonophore Metabolic labeling studies of cerebellar neurons exposedtoaheatshockof42.5%for30-180minshowed marked induction of proteins of approximately 220, 110, 90, 72, 47, and 32 kd (Figure IA). Other than the 32 kd band, which increased with the latertime points, the signal for this group of proteins was maximal after 60 min of heat shock. The optimal time for induction varied slightly between experiments, presumably due tosmall butcritical differences in incubation temperatures. However, maximal induction of HSPs substantially preceded the appearance of thermal toxicity, which was not significant until at least 3 hr of exposure to 42.5V (data not shown). Consistent with prior observations in mammalian cells (Mizzen and Welch, 1988), when neurons were labeled in the presence of [3H]leucine instead of [35S]methionine, an increase in a 28 kd protein was also observed. lmmunoblot analysis using an anti-HSP72 monoclonal antibody verified that HSP72 was one of the major proteins induced by heat shock (Figure 16). Furthermore, immunocytochemistry confirmed that HSP72 expression occurred in the majority of neurons in each dish, rather than a subpopulation of neurons or other cells (e.g., astrocytes; Figure 2). Since prior investigations of the heat shock response have shown that the classic HSPs can be induced also by certain heavy metals, we tested the effects of sodium arsenite on the neuronal cultures. As shown in Figure IA, a number of the HSPs, including those of 90, 72, and 32 kd, were increased following treatment with 50 or 100 W M sodium arsenite. When neuronal cultures were treated with the calcium ionophore A23187, metabolic labeling indicated an increase in expression of proteins of 78 and 94 kd (data not shown). These are likely to be the glucoseregulated proteins (GRPs) that have been described previously (Welch et al., 1983). Analysis of radiolabeled neuronal proteins by twodimensional PAGE confirmed the induction of classic stress proteins by heat shock (Figure 3). Prominent inductions were seen for proteins migrating at the expected molecular weight and isoelectric point for 110,72, and 58 kd HSPs (Welch etal., 1989). In addition, a protein migrating at approximately 50 kd and pl 5.6 was markedly induced by heat shock(Figure3, labeled E). A 90 kd protein (HSP90) was also induced by heat shock, but generally migrated along a broad isoelectric width under our electrophoresis conditions.

4

21 5

--Figure 1. The Induction of Stress Proteins Shock or Sodium Arsenite

in Neurons

by Heat

(A) One-dimensional PAGE analysis of proteins metabolically labeled with [‘Hlleucine. Cells were exposed to 42.5”C for 30-180 min, or to 50 or 100 uM sodium arsenite for 1 hr. Proteins were harvested 6 hr after the start of the experiment. Each lane was loaded with a sample volume equivalent to 200,000 cpm, and the gel was exposed to autoradiograph film for 7 days. Induced proteins of approximate 220, 110, 90, 72, 47, 32, and 28 kd are denoted by the arrows on the right. Molecular weight standards are indicated on the left. (B) lmmunodetection of HSP72 in neurons exposed to 60-180 min of heat shock. Approximately 30 ug of total protein was loaded on each lane. Equivalent protein loading of lanes was verified by Coomassie blue staining on a duplicate gel as well as the post-transfer gel (in which the higher molecular weight proteins were still visible). The first lane shows the presence of HSP72 in heat-shocked HeLa cells. Results shown in (A) and (B) are representative of at least 3 experiments.

Glutamate Does Not induce a Classic Stress Response in Cerebellar Granule Cells Since the pure cultures of cerebellar granule cells could express a classic heat shock response and expressed the GRPs following exposure to a calcium ionophore, we next studied how these cells would respond to treatment with the excitatory amino acid

Stress Proteins in Cultured 1055

Neurons

A

Figure 2. lmmunocytochemical

Detection

of HSP72 in Neurons

following

Heat Shock

but Not Exposure

to Glutamate

lmmunocytochemistry using a monoclonal antibody against HSP72 was done on cultured neurons 24 hr after control conditions at 37OC (A), heat shock at 42.5OC for 1.5 hr (B), or treatment with 250 uM glutamate for 6 hr (C). HSP72 immunoreactivity was evident only following the heat shock. Treatment of cultures with 5 m M glutamate also failed to induce HSP72 immunoreactivity. Results are representative of 2-3 culture dishes for each condition in 2 separate experiments. Bar, 50 pm.

Nonetheless, in multiple experiments that utilized one- and two-dimensional PAGE of pulse-labeled proteins and immunoblot analysis for HSP72, we were unable to identify any consistent changes in HSPs or GRPs following exposure of the cells to various concentrations of glutamate (Figure 4; Figure 5). Furthermore, immunocytochemistry of cells exposed to 250 uM or 5 m M glutamate showed no evidenc:e of HSP72 expression (Figure 2). These observations suggest that, at the very least, glutamate does not induce a classic heat shock response in cerebellar granule cells.

glutamate. Prior reports have shown that glutamate, N-methyl-o-aspartate (NMDA), and other excitatory amino acid receptor agonists can be toxic to cerebellar granule cells, although there is substantial variability in the dose response depending upon culture conditions (Cox et al., 1990; Didier et al., 1990; Lysko et al., 1989; McCaslin and Morgan, 1987). Preliminaryexperiments with our cultures, in which both the magnesium and glucose concentrations were maintained in the normal range (0.8 m M and 7.5 mM, respectively), showed the cells were injured by glutamate concentrations between 100 uM and 10 m M (see Figure 6).

Figure 3. Two-Dimensional PAGE Analysis of Proteins from Heat-Shocked Neurons

Heat Shock

Control ‘0

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Cultured ceils were metabolncally labeled with [?S]methionine and harvested after 6 hr. Controls were maintained at 37V, and heat-shocked cells were exposed to 42.5OCforl hratthe beginningofthemetabolic labeling period. Each gel was loaded with 400,800 cpm, treated with EnHance (New England Nuclear), and exposed to film for 10 days. The molecular weights of known proteins are identified on the right. Labels indicate thefollowing: A, HSPIIO; B, HSP72; C, HSP73; D, HSP58; E, 50 kd stress protein. Results are representative of 3 experiments.

Nl?UKNl 1056

Glutamate

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50 Figure 5. Comparison of Specific Groups of Stress Proteins Neurons after Heat Shock or Exposure to 1 m M Glutamate Hela

Figure 4. One-Dimensional PAGE Indicates Does Not Induce an Obvious Stress Protein tured Neurons

That Glutamate Response in Cul-

Cells were harvested after exposure to different concentrations of glutamate for 6 hr. The top panel shows one-dimensional PAGE analysis of proteins labeled with [‘5S]methionine during the glutamate exposure. Each lane was loaded with 2 x IO5 cpm of radiolabeled protein, and the gel was exposed to autoradiograph film for 7 days. Total TCA-precipitable counts were not significantly different between control and 1 m M glutamate cultures and were approximately 60% lower in 10 m M glutamate cultures. There are no obvious differences in labeled proteins from thecontrol versus theglutamate-exposed cells. The bottom panel shows an immunoblot for HSP72 of protein samples taken from parallel, nonlabeled cultures. Although HSP72 was easily detectable in heat-shocked HeLa cells, there was no HSP72 immunoreactivity in neurons treated with glutamate, even when the autoradiograph was overexposed. Identical results were obtained with glutamate concentrations of 100, 250, 500, and 750 uM.

The Heat Shock Response Confers Thermo-Tolerance and Protection from Excitotoxicity Studies of other in vitro systems have demonstrated that the induction of HSPs is associated with thermotolerance, i.e., a relative resistance of cells to subsequent heat-induced injury (Welch, 1987). We observed this same property in the cerebellar granule cells when they were first exposed to 42.5OC for 1 hr followed by exposure to the normally toxic conditions of 45OC for 30 min (data not shown). We then asked whether the induction of HSPs by mild heat shock mightaffectthesusceptibilityoftheneuronstosubsequent exposure to glutamate. Neuronal cultures were heated to 42.5V for 1.5 hr (which caused induction of HSPs but no toxicity as measured by lactate dehy-

in

Cerebellar granule cell cultures were treated with 1 m M glutamate and concurrently radiolabeled with [‘5S]methionine as described in Figure 3. Cells were harvested and analyzed by two-dimensional PACE.Thefiguredisplaysregionsoftheautoradiograph containing stress proteins in the range of 110, 70, 60, and 50 kd (see Figures 1 and 3). Stress protein inductions are seen only with heat shock (arrowheads). Exposure to 10 m M glutamate was similarly negative for stress protein induction (data not shown). Results are representative of experiments repeated 3 times.

drogenase [LDH] release and trypan blue exclusion), returned to 37OC for 14 hr, and then exposed to a range of glutamate concentrations for 6 hr. Cell viability was assessed 18 hr !ater. The induction of HSP72 was measured by immunoblotting to verify that the heat shock was effective in inducing a stress protein response, and the specificity of the glutamate-mediated toxicity was confirmed by treating some of the cultures with the noncompetitive NMDA receptor antagonist MK.801. As shown in Figure 6, there was a substantial reduction in the toxicity of glutamate in the heat-shocked cells compared with controls. Although there were differences in the extent of this protective effect as determined by trypan exclusion versus LDH release, the protection was consistently observed over a wide range of glutamate concentrations in multiple experiments. For example, at 1 m M glutamate, a prior heat shock resulted in a 26% + 12% (k SD) reduction in LDH release (n = 4, p < 0.02 by Student’s t test) and a 67% zt 12% decrease in the percentage of trypan-positive cells (n = 4, p < 0.001). The protective effect of heat shockwas overcomewith higher glutamate concentrations (5 and 10 mM). Discussion Our findings demonstrate that cultured neurons are capable of expressing classic stress protein responses following exposure to heat shock, sodium arsenite,

Stress Proteins in Cultured 1057

Neurons

Figure 6. Protective Effect of Prior Stress Protein Induction on Glutamate Toxicity

0

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Cerebellar granule cells were exposed to glutamate at the indicated concentrations for 6 hr with or without prior stress protein induction as described in Results. Some cultures were exposed to glutamate in the presence of the noncompetitive NMDA antagonist, MKBOI. Twenty-four hours after exposure to glutamate, cellular toxicity was assessed by measurement of ILDH release (A)ortrypan blueexclusion (B). Datashown are the average of 2 culture dishes at each concentration and are representative of the protective effect of heat shock observed over a range of elutamate concentrations in 4 separite experiments. HSP72 induction was verified by Western blot analysis of cell culture lysates at the onset of glutamate exposure (14 hr after heat shock) (0

MK-801

Glutamate Concentration (PM)

and a calcium ionophore. The spectrum of stress proteins was very similar to what has been described in non-CNS cells. For example, the main pattern of HSP expression following heat shock in our cells appeared nearly identical to the HSPs induced in rat fibroblast, hamster kidney, Chinese hamster ovary, and HeLa cells, as described by Mizzen and Welch (1988). This further substantiates that the stress protein response is highly conserved and, in general, ubiquitousamong a wide range of tissue types and organisms. Ourobservationscontrastwiththosefromtwostudies that have looked at HSP induction in primary culturesof neuronsorastrocytes.Marinietal.(1990)studied cultures enriched for either cerebellar granule cells or astrocytes, as well as mixed cultures. Using immunocytochemistry, they were unable to detect HSP72 expression in neurons following heat stress at 42OC, despite finding HSP72 induction in astrocytes under similar conditions. Nishimura et al. (1991) also investigated HSP expression in cultured cortical neurons and astrocytes. Although one- and two-dimensional PAGE identified the induction of HSP72 (by exposure to 43OC-45V for IO-15 min) in both cell types, there was no significant HSP72 immunoreactivity in neurons when tested with the same monoclonal antibody (C92) used in our experiments. Our ability to detect neuronal HSPs, as well as HSP72 immunoreactivity, was likely related to our use of multiple time points for heat exposure and our empiric finding that heat shock at 42.0°C was generally not sufficient to induceHSPsinourcultures(datanotshown),whereas temperatures of 42.5°-43.00C consistently did so. Thus, small differences in experimental temperature control and exposuretimes could produce significant changes in HSP induction. Surprisingly, we did not detect any induction of HSP72 or other stress proteins when our cerebellar

granule cell cultures were exposed to glutamate. It is unlikely that this lack of induction of classic stress proteins was related to a general impairment of protein synthesis for the following reasons: we studied a wide range of glutamate concentrations that included minimally toxic doses; the total incorporation of [3sS]methionine during metabolic labeling was similar between control and 1 m M glutamate cultures; and treatment with 250 uM glutamate, which caused some injury but left many viable cells, did not produce any HSP72-immunoreactive neurons (Figure 2). Experiments with MK-801 showed that the glutamate-induced neuronal injury was probably mediated by the NMDA receptor, as has been shown by other investigators with both in vivo and in vitro models (Hahn et al., 1988; Michaels and Rothman, 1990). Since in vivo studies have demonstrated that HSP72 is expressed in neurons following various forms of presumed excitotoxic injury, our observations suggest that factors other than excitatory amino acids may contribute to the cellular pathophysiology of these insults. For example, ischemia produces hypoxia, acidosis, and increases in free radical production, in addition to increases in extracellular glutamate (Choi, 1988). In this regard, Benjamin et al. (1990) have recently shown that hypoxia can directly activate the heat shock transcription factor that regulates induction of HSP72 by heat and other stress factors. Excessive NMDA receptor activation is thought to be toxic to neurons as a direct result of prolonged and large magnitude fluxes of calcium into the cell (Choi, 1988). One might expect, therefore, that glutamate should particularly induce the GRPs, since these proteins have been characterized as responding to calcium ionophores. However, in multiple experiments using both one- and two-dimensional PACE, we did not observe an increase in grps following exposure of

Neuron 1058

neurons to glutamate. This lack of GRP induction by glutamate may be related to the suggestion by Drummond and coworkers (1987) that CRP induction following treatment with calcium ionophores is caused by a depletion of intracellular stores of calcium rather than an influx of extracellular calcium. The last experiments presented here provide evidence for a functional role of the heat shock response in CNS neurons. Induction of HSPs was associated with thermo-tolerance, similar to prior observations in nonneural cells. More importantly, we found that induction of HSPs was associated with protection from subsequent glutamate-mediated toxicity across a wide range of glutamate concentrations. It remains to be shown whether this protection was directly related to HSPs. Nonetheless, our results provide the most direct evidence to date that the modulation of HSP expression by relatively nontoxic inducers may be an additional strategy for ameliorating neuronal injury related to a variety of CNS diseases. Experimental

Procedures

Primary Cultures Primary cultures of cerebellar granule ceils were prepared as described by Yu et al. (1986) and used at age 11 days. Neuronal characteristicsof thesecultures have been previously verified by immunocytochemistry using antiserum against neuron-specific enolase (Marangos and Schmechel, 1987). Less than 3% of the cells exhibited immunoreactivity with an antibody directed against glial fibrillary acidic protein. More than 95% of the cells were viable based on their ability to exclude trypan blue dye. Conditions for Inducing Stress Proteins Heat Shock Cells were taken from their normal incubating temperature (37OC) and moved immediately onto large, sand-filled trays within an incubator set at 42.S°C (for inducing HSPs) or 45OC (for heat toxicity). Preliminary experiments using temperatures ranging from 42.0°C to 43.0°C showed that 42.5”C was the minimum temperature for reproducibly inducing a heat shock response. Heat conditions were monitored via a mercury thermometer placed directly in the sand. Sodium Arsenite A 10 m M stock solution of sodium arsenite was made with sterile PBS, and aliquots were added directly to culture media to yield final concentrations of 50 or 100 PM. Calcium tonophore The calcium ionophore A23187 (Sigma Chemical Co.) was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 100 mM. This stock solution was diluted further with DMSO to 208 PM just prior to use, and aliquots were added directly to culture media to yield final concentrations of 1.0-10 NM. Equivalent volumes of pure DMSO were added to control cultures. Fxcitotoxicity L-Glutamate (Sigma Chemical Co.) was mixed at a concentration of 208 m M in PBS, and the pH was adjusted to 7.4 using 1 N NaOH. This was added directly to culture media to yield final concentrations ranging from 180 uM to 10 mM. To test for the receptor specificity of glutamate toxicity, the excitatory amino acid receptor antagonist MK- 801 (prepared as a 20 m M stock in sterile ethanol; final concentration 1 PM) was added to some cultures. Measurements of Cell Toxicity Trypan Blue Stainirtg Trypan blue stock solution (0.4%) was added directly to tissue culture dishes to achieve a final concentration of 0.02%, and the cells were incubated for 10 min. Injured (blue) and viable

(non-blue) cells rn four to five 208x fields were counted via phase-contrast microscopy. All cell cultures for a given experiment were prechecked to verify that they were of equal confluence. LDH Activity LDH released from damaged cells was determined by taking aliquots of culture medium; detached cells and debris were removed by centrifugation at 3808 x g. The remaining cellular LDH was obtained by lysing cell monolayers with a 0.2% Triton X-100, PBS solution. LDH activity in 50 ul samples of medium or cell lysates was measured using an LDH assay kit (Sigma) and procedures specified by the supplier; changes in absorbance per min at 348 nm were measured by spectrophotometry at room temperature. The percent LDH release was defined as the ratio of LDH activity in the medium and total activity (i.e., medium plus cell lysates) per dish. Metabolic Labeling Cells were labeled for 6 hr with [?j]methionine (50 PCi per culture dish; DuPont, New England Nuclear) in methioninefree, serum-containing medium. (In some experiments labeling was done with [‘Hlleucine and leucine-free medium.) After labeling, the medium was removed and the cells were washed 3 times with cold PBS. For one-dimensional PAGE, the cells were then lysed in Laemmli sample buffer(200 ul for 30 m m plates; Laemmli, 1970)containing leupeptin, aprotinin, and soybean trypsin inhibitor at 20 pg/ml each. RNAase A and DNAase I were then added to cell lysates at concentrations of 50 uglml and 100 uglml, respectively. Samples were heated to85’C for 5 min, IO ul aliquots were removed for determination of trichloroacetic acid (TCA)-precipitable counts, and remaining samples were kept frozen at -70°C until further use. For two-dimensional PAGE, the cells were lysed with 9 M urea, 4% (v/v) Nonidet P40 as described by Q’Farrell (1975) and processed as above, but without heating to 8!?‘C. One-and Two-Dimensional PAGE OneDimensional PAGE Sample volumes with equal TCA-precipitable counts, ranging from 125,000 to 400,808 cpm for each gel, were loaded onto 1.5 m m thick gels composed of a 3% acrylamide stacking gel and a 10% acrylamide running gel. Electrophoresis was carried out at 50 V overnight. Gels were then fixed with a 50% methanol, 10% acetic acid solution for 1-2 hr, dried, and exposed to Kodak XAR-5 film for 1-3 weeks. Two-Dimensional PAGE Proteins were analyzed by two-dimensional PAGE according to Hochstrasser (Hochstrasser, Harrington et al., 1988) using a pH gradient of 4.8-7.2 and 10% SDS-polyacrylamide gels. Identical amounts of radioactivity (1.5 x 105 cpm) were loaded for each sample. Multiple (12-20) second-dimension gels were run simultaneously using a Protean II Multicell (Bio-Rad) or Iso-Dalt (Hoeffer Scientific) apparatus. Duplicate gels were run on each sample within a given experiment, thus giving 4-6 individual gels for each experimental treatment. Following electrophoresis, gels were treated with EnHance (New England Nuclear), dried, and then exposed to X-ray film that had been pm-flashed to improve linearityofthefilm response(Laskeyand Mills, 1975). Qualitative analysis of autoradiographs was done by visual inspection. Indicated proteins were identified by characteristic molecular weights and isoelectric points. lmmunoblot Analysis Protein samples were prepared as described above for onedimensional PAGE, except that the proteins were not radiolabeled. Protein lysates were then subjected to immunoblot analysis as described previously (Miles et al., 1990). A mouse monoclonal antibody directed against HSP72 (C92) was kindly provided by Dr. W. Welch. lmmunocytochemistry Culture dishes were washed twice with cold PBS and fixed with -20°C methanol. Cells were then gently washed in 0.1 M Tris buffer (pH 7.6) 3 times for 5 min each, 1.0% hydrogen peroxide

Stress Proteins in Cultured 1059

Neurons

in Tris buffer for 10 min,Tris buffer for 5 min, Tris A (0.05% Triton X-100 in Tris buffer) for 10 min, Tris B (0.05% Triton X-100 and 0.005% bovine serum albumin in Tris buffer) for 10 min, 10% normal horseserum inTrisBfor30min,TrisAforlOmin,andTris B for 10 min. The cells were then incubated with the monoclonal antibody C92 diluted I:2000 in Tris B overnight at 25OC. The following day, the cells were washed in Tris A and Tris B for 10 min each, incubated in biotinylated horse anti-mouse IgG (Vector Labs, Burlingame, CA) at I:400 dilution in Tris B for 2.5 hr, washed in Tris A and Tris B for 10 min each, incubated in avidin-biotin-peroxidase (ABC kit from Vector Labs) at I:1000 dilution in Tris B for 2 hr, washed in Tris buffer 3 times for 5 min each, and then exposed to a mixture of 0.5 mg/ml diaminobenzidine tetrahydrochloride, 3 mglml glucose oxidase, 200 mg/ml ammonium chloride, and250 mglml o-(+)-glucose. Cultureswere reacted until optimal staining intensity was achieved (60 min) and then washed and stored in Tris buffer.

This work was supported in part by grants NS01424 (D. H. L.), NS 14543 (P. H. C.), and NS 25372 (P. H. C.) from the National Institutes of Health, AA00118 (M. F. M.) from the NIAAA, and a grant from the American Epilepsy Society (D. H. L.). We wish to thank Julie Archer, Lillian Chu, SylviaChen,Thomas McCabe, and Winnie Chin for excellent technical assistance and Drs. David Greenberg and Daria Mochly-Rosen for their critical review of the manuscript. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

May 21, 1991; revised

September

Gonzalez, M., Shiraishi, K., Hisanaga, K., Sager, S., ,Wandabach, M., and Sharp, F. (1989). Heat shock proteins as markers of neural injury. Mol. Brain Res. 6, 93-100. Gonzalez, M., Lowenstein, D., Fernyak, S., Hisanaga, K., Simon, R., and Sharp, F. (1991). Induction of heat shock protein 72-like immunoreactivity in the hippocampal formation following transient global ischemia. Brain Res. Bull. 26, 241-250. Cower, D., Hollman, C., Lee, K., and Tytell, M. (1989). Spinal cord injuryand the stress protein response. J. Neurosurg. 70.605-611. Hahn, J. S., Aizenman, E., and Lipton, S. A. (1988). Central mammalian neurons normally resistant to glutamate toxicity are made sensitive by elevated extracellular Ca2+: toxicity is blocked by N-methyl-o-aspartate antagonist MK-801. Proc. Natl. Acad. Sci. USA 85, 6556-6560. Hochstrasser, D. F., Harrington, M. C., Hochstrasser, A. C., Miller, M. J., and Merril, C. R. (1988). Methods for increasing the resolution of two-dimensional protein electrophoresis. Anal. Biochem. 773, 424-435. Jacewicz, M., Kiessling, M., and Pulsinelli, gene expression in focal cerebral ischemia. Metab. 6, 263-272.

W. (1986). Selective J. Cereb. Blood Flow

Kiessling, M., Dienel, G., Jacewicz, M., and Pulsinelli, W. (1986). Protein synthesis in postischemic rat brain: a two-dimensional electrophoretic analysis. J. Cereb. Blood Flow Metab. 6,642-649. Kirino, T., Tsujita, Y., and Tamura, A. (1991). Induced tolerance to ischemia in gerbil hippocampal neurons. J. Cereb. Blood Flow Metab. 11, 299-307. Kitagawa, K., Matsumoto, M., Tagaya, M., Hata, IR., Ueda, H., Niinobe,M., Handa, N., Fukunaga, R., Kimura, K., Mikoshiba, K., and Kamada, T. (1990). ‘Ischemic tolerance’ phenomenon found in the brain. Brain Res. 528, 21-24.

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