Stress-induced Inhibition of Protein Synthesis

The Journal

of Neuroscience,

May

1993,

73(5):

1830-1838

Stress-induced Inhibition of Protein Synthesis Initiation: Modulation of Initiation Factor 2 and Guanine Nucleotide Exchange Factor Activities following Transient Cerebral lschemia in the Rat Bing

Ren Hu and Tadeusz

Wieloch

Department of Neurobiology, Laboratory for Experimental Brain Research, Lund Hospital, Lund University, S 221 85 Lund, Sweden

Neuronal protein synthesis is severely depressed following stress such as heat-shock, hypoxia, and hypoglycemia. Following reversible cerebral ischemia, protein synthesis is transiently inhibited in ischemia-resistant areas, but persistently depressed in vulnerable brain regions. Eukaryotic initiation factor 2 (elF-2) activity, that is, the formation of the ternary complex elF-2.GTP initiator 35S-Met-tRNA, a ratelimiting step in the initiation of cellular protein synthesis, was studied in the rat brain during and following 15 min of transient global cerebral ischemia. At 30 min and 1 hr of reperfusion, a general decrease of elF-2 activity by approximately 50% was seen in the postmitochondrial supernatant (PMS). In the relatively resistant neocortex and CA3 region of the hippocampus, the elF-2 activity returns to control levels at 6 hr of reperfusion, but remains depressed in the vulnerable striatum and the CA1 region. Similarly, the activity of the guanine nucleotide exchange factor (GEF), which catalyzes the exchange of GTP for GDP bound to elF-2, a crucial step for the continued formation of the ternary complex, is transiently reduced in neocotiex but persistently depressed in striatum. The postischemic decrease in elF-2 activity is further attenuated by agarose-bound alkaline phosphatase, and mixing experiments revealed that a vanadate-sensitive phosphatase may be responsible for the depression. Addition of partially purified GEF to PMS from postischemic neocortex restored elF-2 activity to control levels. We conclude that ischemia alters the balance between phosphorylation and dephosphorylation reactions, leading to an inhibition of GEF and a depression of ternary complex formation. The persistent inhibition of GEF and ternary complex formation in areas vulnerable to ischemia may be due to factors causing cell death.

[Key words: ischemia, phosphatase, protein synthesis, initiation factor 2, guanine exchange factor, neuronal death, phosphorylation] A wide range of conditions such as heat-shock, amino acid or glucosedeprivation, chemicalstress,and viral infection depress global cellular protein synthesisrate (Pain et al., 1980;Siekierka Received Jan. 22, 1992; revised Sept. 17, 1992; accepted Oct. 22, 1992. We thank Margareta Svejme for the skillfully performed animal experiments. This work was supported by Swedish Medical Research Council Grant 06844, The Medical Faculty at Lund University, The Segerfalk Foundation, The Crafoord Foundation, and The Swedish Association for Medical Research. Correspondence should be addressed to Dr. Tadeusz Wieloch at the above address. Copyright 0 1993 Society for Neuroscience 0270-6474/93/l 3 1830-09$05.00/O

et al., 1985;Scorsoneet al., 1987).The influenceof stress,caused by transient cerebral ischemia, on protein synthesis has been intensively studied. During the reperfusion phasefollowing a brief period of cerebral ischemia, polyribosomes disaggregate (Cooper et al., 1977; Petit0 and Pulsinelli, 1984a,b;Munekata et al., 1987; Deshpandeet al., 1992) and protein synthesis,as measuredby the incorporation of radioactive amino acids into brain proteins, is depressed(Dienel et al., 1980, 1985; Bodsch et al., 1985;Thilmann et al., 1987;Araki et al., 1990;Widmann et al., 1991). In areasvulnerable to ischemia, such as the CA1 region of the hippocampus,protein synthesisis persistently inhibited, while it is transiently depressedin the hippocampal CA3 region, dentate gyrus, and neocortex, areasrelatively resistant to ischemia. The correlation between selective vulnerability to ischemiaand the persistentdepressionof protein synthesissuggests a causalrelationshipbetweeninhibition of protein synthesisand neuronal death (Bodschet al., 1985). The mechanismsunderlying the postischemicinhibition of protein synthesis are still virtually unknown. However, neuronal maturation, development, and survival are intimately associatedwith the presenceof growth factors, which through the activation of their cell surface receptors affect among others the translation of mRNA via phosphorylation-dephosphorylation reactions (Morley and Thomas, 1991). Cellular protein synthesisis commonly divided into an initiation, an elongation, and a termination step. It is generally recognizedthat the rate of polypeptide synthesisis regulatedat the initiation stepby the eukaryotic initiation factors (eIFs), in particular eIF-2 and eIF-4 (Nygard and Nilsson, 1990; Hershey, 1991; Morley and Thomas, 1991; Rhoads, 1991). Initiation factor 2 is believed to modulate the overall protein synthesis rate, while eIF-4 is thought to regulate the selectionand translational efficiency of different mRNA species.Initiation of protein synthesisconstitutes a seriesof intricate processesstarting with the formation of a complex betweenGTP and eIF-2 that when binds the initiator Met-tRNA, Met-tRNAi, to form a ternary complex (eIF-2.GTP,Met-tRNAi) (Ochoa, 1983; Pain, 1986) often referred to as eIF-2 activity. The ternary complex associateswith ribosomal subunits, additional eIFs, and the mRNA in a seriesof interactions leading to the formation of the mRNA’80S ribosomal complex. Concomitantly, GTP is hydrolyzed and eIF-2.GTP is released.The GDP bound to eIF-2 must be exchangedfor GTP to regenerateeIF-2.GTP for another round of initiation. Since eIF-2 has higher affinity for GDP than for GTP, the exchange processis catalyzed by the guanine nucleotide exchangefactor (GEF) (Proud, 1986). Recycling of eIF-2 is essentialfor a continued initiation of protein

The Journal

synthesis, and the exchange of GDP for GTP is a rate-limiting step. Two regulatory mechanisms for this process have been proposed. One involves the phosphorylation of the a-subunit of eIF-2, which in its phosphorylated state binds GEF in a stable complex with GDP (Proud, 1986; Scorsone et al., 1987; Clemens, 1989). The other mechanism involves the modulation of GEF activity by phosphorylation-dephosphorylation reactions (Dholakia and Wahba, 1988; Tuazon and Traugh, 1991). The objective this study was to investigate whether severe stress such as cerebral ischemia alters the activity of brain eIF-2 and GEF, and whether particular protein kinases or phosphatases are involved in these processes.

Materials and Methods The chemicals were obtained from the following sources: L-YS-methionine and 1251-anti-mouse IgG from Amersham (Amersham, UK); tRNA of rabbit liver type XII, aminoacyl-tRNA synthetase from Escherichia co/i, phosphoenolpyruvate (PEP), pyruvate kinase (PK), ATP, GTP, benzoylated naphtoylated DEAE (BND)-cellulose, leupeptin, and 5[Nmorpholinolpropanesulfonic acid (MOPS) from Sigma (St. Louis, MO); nitrocellulose (NC) filter (0.2 pm, type HA) from Millipore. All other chemicals were of analytical or reagent grade. The monoclonal antibody against eIF-2o was kindly provided by Professor E. C. Henshaw (Rochester University, Rochester, NY). Induction of ischemia. The two-vessel occlusion model of global cerebral ischemia according to the method of Smith et al. (1984), as modified by Gustafson et-al. (1989), was used. The experiments were aooroved -rr-~~ bv -, the ethical committee at Lund University. Male Wistar rats (350-400 gm) (Mollegaard A/S, Copenhagen, Denmark) were used. Before surgery the animals were fasted overnight with free access to tap water. Anesthesia was induced by placing the rat in a jar with 3% isoflurane in a mixture of oxygen/nitrous oxide (30/70%). Following intubation with a plastic tube, they were artificially ventilated by a rodent respirator (7025 Rodent Ventilator, Ugo Basile Biological Research Apparatus, Comeno, Italy). Anesthesia was maintained with l-2% isoflurane in the oxygen/nitrous oxide (30/704/o) gas mixture. An external jugular vein catheter was then inserted, and positioned in the superior caval vein. A tail artery catheter and vein catheter were also inserted to allow blood sampling, arterial blood pressure recording, and infusions of drugs. The arterial blood pressure was measured and recorded continuously until the rats could be extubated. In all animals both common carotid arteries were exposed and encircled by loose ligatures. Arterial blood samples (200 hl) were collected 15 min before ischemia and 15 min postischemia to measure blood gases and blood glucose, If blood gases could not be corrected (P=O, > 90 mm Hg, PaCO, 3545 mm Hg, pH 7.35-7.45) by adjustments of the rodent ventilator, or if the blood glucose was ~4 mmol/liter, the rats were excluded from the study. At the end of the surgery bipolar EEG electrodes were inserted into the temporal muscles and the EEG activity was recorded (Mingograf 34, Elema-Schonander, Stockholm, Sweden) every 5-10 min before ischemia, continuously during the ischemic insult, and intermittently in the postischemic period. These electrodes were removed once postischemic EEG activity recovered. At the beginning of a 30 min steady-state period prior to induction of ischemia, the inspired isoflurane concentration was decreased to 0.5% and 150 III/kg heparin was administered intravenously. Vecuronium (Organon Teknika, Boxtel, Holland), a muscle relaxant, was given as a bolus dose of 0.7 mg followed by an intravenous infusion of 3 mg/hr. Blood was withdrawn via the jugular catheter to a mean arterial blood pressure (MABP) of 50 mm Hg and both carotid arteries were clamped. Blood pressure was maintained at 50 mm Hg during the ischemic period by withdrawing or infusing blood through the jugular catheter. The beginning of the ischemic insult was defined as the time of onset of isoelectric EEG at an MABP of 50 mm Hg. At the end of ischemia the clamps were removed and the blood reinfused through the jugular catheter, followed by 0.5 ml of 0.6 M sodium bicarbonate. In all experiments, temperature was monitored before, during, and following ischemia (15 min of reperfusion), using thermistors placed in the rectum and subcutaneously under the scalp. Temperature control was accomplished with the aid of a heating pad and kept at approximately 37°C. Animals to be killed immediately at the end of &hernia or following 1 hr reperfusion were left on isoflurane anesthesia and the brain was frozen in situ with liquid nitrogen. Both vecuronium

of Neuroscience,

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1993,

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and isoflurane, administered to the animals that were allowed to recover for 6 hr postischemia, were discontinued at the end of ischemia. Following the removal of the external jugular vein catheter, all wounds were sutured. Within 45 min after the ischemic insult the animals resumed adeouate soontaneous breathing and the endotracheal tube could be removed. The animals were transferred to a cage where they received supplementary oxygen. Animals killed at 6 hr postischemia were reanesthetized on isoflurane, tracheostomized, and artificially ventilated on isoflurane. The brains were then frozen in situ. The brains were dissected out at - 17°C. Neocortex (parietal cortex) was sampled rostra1 to 3.5 mm from bregma. Striatum was sampled from both hemispheres. The CA1 region and CA3 region plus dentate gyrus of the hippocampus were sampled at the dorsal aspect of the hippocampus. Subcellular fractionation. Ten to twentv millierams of frozen tissue were sonicated for 3 x 10 set bursts at an output setting of 30 (Ultrasonic Homogenizer, Cole-Parmer Instrument), in a homogenization buffer containing 50 mM MOPS (pH 7.4) 0.5 mM magnesium acetate, 100 mM KCl, 1 mM dithiothreitol (DTT), 0.1 mM EDTA, 50 mM NaF, and 0.1 mg/ml leupeptin. In the mixing experiments or the experiments employing alkaline phosphatase treatment, NaF was omitted the homogenization buffer. Following homogenization a postmitochondrial supematant (PMS) was obtained by centrifugation in a Sorvall SE12 rotor, 15,000 rpm for 15 min at 4°C. The fractions were stored at - 70°C in aliquots or used immediately. The samples were allowed to thaw only once, since repeated freezing-thawing cycles inactivate eIF-2. The protein concentration was determined by the method of Lowry et al. (1951). Synthesis of ‘JS-Met-tRNAi. Initiator tRNA, YS-Met-tRNAi, was svnthesized bv lieatine rat liver tRNA with L-YS-methionine (500 mCi/ mmol) in presence OF E. coli synthetase according to the methods of Smith and Henshaw (1975). The incubation buffer (buffer A) contained 100 mM Na-cacodylate, pH 7.0, 10 mM KCl, 2 mM DTT, 10 mM magnesium acetate, and 2.5 mM ATP. The reaction was initiated by addition 140 U of E. coli aminoacyl-tRNA synthetase (35 U/pi) to 1 ml of buffer A containing 2 mCi of LJ5S-methionine and 50 U of rat liver tRNA. Following 10 min of incubation at 37°C 15 ml of ice-cold buffer B (10 mM acetate buffer, pH 4.5, 125 mM NaCl) was added to stop the reaction and the mixture was applied on a BND-cellulose column (1 x 2 cm) preequilibrated with buffer B. The column was washed thoroughly with buffer B (1 ml/min, 30-40 ml) until A,,, was back to baseline and was then washed with 0.5 M NaCl in buffer B with a flow rate of 0.1 ml/ min. Fractions (2 ml) were collected and 2 ~1 was taken for measurement of specific eIF-2 activity, Fractions with highest specific activity were pooled, precipitated with 2 vol ofice-cold ethanol (99%), and centrifuged (20,000 rpm; 80,000 x g) for 15 min at 2°C in a Beckman 28.1 rotor. The precipitate was washed with 70% and 90% ethanol, respectively, and finally dissolved in 2 mM DTT solution to a specific radioactivity of 15,000 cpmlpl. Aliquots of 100-200 ~1 were stored at -80°C until further use. Measurement of ternary complex .formution. The ternary complex formation in PMS was measured as the GTP-dependent retention of YS-Met-tRNA as a temarv comulex, GTP.eIF-2.‘S-Met-tRNAi, on NC filters according to the methods of Wong et al. (1982) with some modifications. In the absence of GTP or a GTP-regenerating pyruvate kinase system in the assay, ternary complex formation is low. The ternary complex formation increases with increasing GTP concentrations, and potassium and magnesium ions are obligatory (Dwyer and Wasterlain, 1980). For our assay conditions we chose an incubation time of 10 min, and a reaction mixture (100 ~1) that contained 50 mM MOPS, pH 7.4, 1 mM DTT, 100 mM KCl, 0.75 mM magnesium acetate, 75 PM GTP, 0.25 mM PEP, 6 U/ml of PK, 150 &ml of BSA, 5 ~1 of YS-Met-tRNAi (0.4 pmol), and 20 ~01% (150 wg protein) of PMS fraction. The assay was linear up to 300 Mg added PMS protein. Following incubation for- 10 min at 3O”c, the reaction was stopped by addition of 2.5 ml ice-cold washinr! buffer consisting of 20 mM MOPS, uH 7.4. 0.1 mM DTT, 100 mM KCl, and 3 mM magnesium acetate, and the solution was immediately passed through an NC filter under mild suction. The filter was washed four times with 2 ml of washing buffer, dried under a heating lamp, and placed in a scintillation vial, and 10 ml of ReadySafe cocktail (Beckman) was added. Radioactivity was counted in a Beckman LS 2800 liquid scintillation counter. Electrophoresis and immunoblotting. Electrophoresis was carried out on a 1.5 mm, 10% SDS-polyacrylamide gel according to the method of Laemmli (1970). The samples (50 pegprotein) were mixed with a solution

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Stress

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of 0.3 M Tris-HCI, pH 6.8, 25% mercaptoethanol, 12% SDS, 25 mM EDTA, 20% glycerol, and 0.1% bromphenol blue (5 x SDS), boiled 2 min, and subjected to gel electrophoresis at a constant current of 20 mA (stacking gel) and 30 mA (separating gel). Following electrophoresis, proteins on the gel were electrotransferred onto NC (Bio-Rad Transblot, 0.2 mm) using the method of Towbin et al. (1979) with a constant current of 200 mA overnight. After transfer the NC was washed once in PBS, washed (2 x 10 iin) in PBS plus 0.1% Tween 20, and then vreincubated with 3% BSA in PBS for 1 hr. It was incubated with a Solution containing monoclonal antibody against eIF-2a in PBS with 3% BSA for 2 hr. Finally, the NC was incubated with 0.5 mCi/ml 12sIanti-mouse IgG for 1 hr. All incubations were carried at room temperature. The NC was placed in a plastic bag and exposed onto Kodak X-Omat film for about 24 hr at -80°C in a cassette with Hi-plus intensifying screen. The protein bands on the film were identified according to the molecular weight or purified eIF-2cu standard run on the same gel. The autoradiograms were scanned with a laser scanner, and the relative density of the eIF-2a bands on the film was calculated and expressed as percentage of control values. The densities were linear with the amount of eIF-2 present within the range measured. Purification of eIF-2. Initiation factor 2 was purified from calf brain according to thk methods of Cales et al. (1988) and Haro and Ochoa t 1979) with some added modifications. Calf brain (500 gm) was obtained irom a local slaughterhouse, where cortex was dissectid out and frozen in liquid nitrogen. Prior to preparation the frozen tissue was crushed into smaller pieces and homogenized in a blender for 3 x 1.5 min with ice-cold homogenization buffer (3 ml/gm tissue). The homogenization buffer consisted of 20 mM Tris-HCI, pH 7.6, 150 mM KCI, 5 mM magnesium acetate, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 0.1 mM EDTA, and 0.125 mM sucrose. The homogenate was centrifuged in Sorvall GSA rotor at 10,000 rpm for 15 min, and the resulting supematant was centrifuged in a Beckman 28 rotor, at 28,000 rpm for 4 hr. The pellets (microsomal fractions) were stored at -80°C or immediately used for next purification step. The microsomal fraction was suspended with 210 ml bf buffer A (20 mM Tris-HCl, pH 7.6, 1 mM DTT. 0.1 mM EDTA. 0.1 mM PMSF. 10% dvcerol) and made uv up to a final KC1 concentration of 0.5 M dy slo