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Selective c‐JUN Expression in CA1 Neurons of the Gerbil Hippocampus during and after Acquisition of an Ischemia‐Tolerant State

Selective c‐JUN Expression in CA1 Neurons of the Gerbil Hippocampus during and after Acquisition... Selective c-JUN Expression in C A I Neurons of the Gerbil Hippocampus during and after Acquisition of an Isc hemia-ToIerant State Clemens Sommer, Peter Gass, Marika Kiessling Department of Neuropathology, University of Heidelberg, D-69120 Heidelberg, Germany Introduction The selective delayed neuronal death of CAI pyramidal cells after transient global ischemia i n the gerbil brain can be prevented by preconditioning with a short sublethal period of ischemia 1 - 7 days prior t o a subsequent, usually lethal ischemia of 5 min duration. Since changes of neuronal gene expression may play a crucial role in this tolerance induction, w e investigated the postischemic expression profile of the fos, jun and Krox transcription factor families. We have previously reported that a single 5 min period of cerebral ischemia does not cause a de novo synthesis of immediate early gene (IEG) encoded proteins in CAI neurons. In the present study, t w o experimental groups of Mongolian gerbils were investigated: one group was subjected t o a single tolerance-inducing 2.5 min period of ischemia by bilateral occlusion of the common carotid artery. The second (combined ischemia) group was subjected t o 2.5 m i n of ischemia, followed b y 5 min of ischemia 4 days later. Postischemic expression of c-FOS, FOS B, c-JUN, JUN B, JUN D and KROX-24 was investigated by in situ hybridization and immunocytochemistry up t o 48 h of recirculation. In contrast t o a single 5 min period of ischemia, 2.5 min caused a postischemic expression of c-JUN protein, but no other IEGs, i n C A I neurons (peak at 6 h). Similarly, a selective but delayed c-JUN expression (peak at 18 h) was observed in animals subjected t o combined ischemia. These results indicate that the induction of an endogenous neuroprotective state in CAI neurons is associated with the activation of a genetic program which involves the expression of specific transcription factors. Received 1 1 January 1995 Corresponding author Dr M Kiessling, Institute of Neuropathology, University of Heidelberg, D-69120 Heidelberg, Germany Tel +49 (6221) 56 2603, Fax +49 (6221) 56 3466 Transient global forebrain ischemia of 5 min duration in the gerbil severely affects energy metabolism in several areas of the central nervous system (CNS), but in the hippocampus only CA1 neurons undergo selective and delayed degeneration [29]. Recently, a number of studies have demonstrated that highly vulnerable hippocampal CA1 neurons both in the gerbil [25, 30, 321 and in the rat [37] can be protected significantly against ischemic damage by prestressing with a short sublethal ischemic period. This phenomenon of ischemia-tolerance induction has also been described in other brain regions [14, 311, and after preconditioning by hyperthermic [6, 331 or systemic oxidative stress in vivo [45]. The molecular basis of tolerance induction is not yet understood. Two studies suggested the possibility that loss of hilar somatostatin neurons by short periods of ischemia [39] or destruction of dentate granule cells by colchicine [23] could confer protection of CAI neurons by triggering a modulation of excitatory neurotransmission in the hippocampal circuitry. Considerable interest, however, has been attracted by several investigations which demonstrate a coincidence of tolerance induction and the expression of a 70 kDa heat shock protein (HSP70) in vivo [14, 30, 37, 501, suggesting a possible role of stress proteins in the acquisition of an ischemia-tolerant state. In analogy, in vitro induction of HSP70 in cultured neurons has been shown to be associated with increased tolerance to glutamate toxicity [38, 481. It is well known that living organisms, when exposed t o sublethal environmental stress such as high temperature, amino acid analogues, heavy metals or inhibition of energy metabolism, respond by intrinsic alterations of the molecular phenotype, preferentially by initiating the synthesis of several so called heat shock or stress proteins (for review see [5, 18, 24, 36, 441). However, there is n o direct evidence linking stress protein expression to tolerance induction. Furthermore, recent data indicate that HSP70 induction after global ischemia in the gerbil predominantly depends o n postischemic hyperthermia [52] and in focal ischemia models even occurs after sham occlusion of the middle cerebral artery [14]. Low molecular Sommer et al: Selective c-JUN Expression in C A I Neurons weight stress proteins, e.g. HSP27, also show a temporal expression compatible with the induction of an ischemia-tolerant state, but the distribution pattern makes it unlikely that they are involved in neuroprotection by preconditioning [26]. To the best of our knowledge, no changes in gene expression in CA1 neurons other than the heat shockfstress response have been reported following prestressing. One other gene family potentially involved is the class of immediate early genes (IEGs) which are also known t o respond to a variety of noxious stimuli, including ischemia [ll, 13, 17, 27, 28, 541. IEGs are thought t o be third messengers in a complex cellular cascade of stimulus-transcription coupling that converts extracellular signals into alterations of cellular functions by regulating target gene expression, such as neurotransmitter or neuromodulator genes [15, 43, 511. In Mongolian gerbils subjected to a single 5 min period of transient global ischemia, irreversibly damaged CA1 neurons exhibit strong transcription of various IEGs [28], but probably due to a severe and persistent inhibition of protein synthesis [3, 53, 551, no translation occurs. In contrast, a short priming ischemic period which is sufficient for tolerance aquisition provides the potential for changes in gene expression to become effective. It causes no appreciable neuronal damage, and is associated with recovery of protein synthesis in CA1 neurons [lo, 421. To study the IEG response during tolerance acquisition, we examined the synthesis of six transcription factors of the fos, jun and Krox families in the gerbil hippocampus after a single tolerance-inducing 2.5 min period of ischemia and various recirculation periods. To examine the effect of tolerance acquisition on IEG expression, a second group of gerbils was subjected t o combined 2.5 min and 5 min of forebrain ischemia 4 d apart. For three representative members of different IEG subclasses (c-fos, c-jun, Krox-24) analysis in the combined ischemia group was performed both at the transcriptional and the translational level. Material and Methods (combined ischemia group). Anaesthesia was induced with a mixture of 30% 02, 700/0 N,O, and 1.5% halothane. In both experimental groups the common carotid arteries were exposed and double clamped with atraumatic Biemer aneurysm clips. In both groups clips were removed and restoration of blood flow was visually verified after 2.5 min. In the combined ischemia group animals were resubjected to a 5 min period of ischemia four days later using the same protocol. Expression of six IEG encoded proteins (c-FOS, FOS B, c-JUN, JUN B, JUN D, KROX-24) were investigated immunocytochemically in both experimental groups after recirculation intervals of 3 h, 6 h, 12 h, 18 h, 24 h and 48 h (n=4 per time point). Control gerbils of both groups were subjected to anaesthesia and all surgical procedures, except clamping of the carotid arteries (n=1 per time point). In addition, baseline levels of IEGs were determined in 3 untreated animals to exclude possible effects due to handling and anaesthesia. Animals were sacrificed by transcardiac perfusion under deep ether anaesthesia. Heparin (90 IU) was given via the left ventricle prior to washout of blood vessels with isotonic saline. Brains were perfusion-fixed with 4 % (w/v) paraformaldehyde solution, removed and postfixed overnight in the same fixative prior to vibratome sectioning. Comparative analysis of representative IEG mRNAs (c-fos, c-jun, Krox-24) was carried out in the combined ischemia group by in situ hybridization using the same postischemic time points as for immunocytochemistry plus one additional earlier time point at 1 h of recirculation (n=3 per time point). Animal experiments. Induction of ischemia tolerance was performed according t o the protocol of Kirino et al. [30] with minor modifications. We employed the bilateral carotid occlusion model as already described in detail in previous publications from our laboratory [28, 531. Experiments were performed on adult male Mongolian gerbils (Meriones unguiculatus, 70-80 g) obtained from Tumblebrook Farms (West Brookfield, MA, USA). In addition to the previously studied standard model of a single 5 min period of ischemia [28], two experimental groups of animals were investigated: one group was subjected to a single tolerance inducing 2.5 min period of ischemia (2.5 min initial ischemia group). The second group was subjected to 2.5 min of ischemia, followed by 5 min of ischemia 4 d later In situ hybridization. For in situ hybridization, brains were rapidly removed and frozen in 2-methylbutane at -30°C. Coronal sections 14 pm thick at the level of the dorsal hippocampus were cut in a cryostat at -2O"C, dried o n poly-L-lysine coated slides and fixed in 4% paraformaldehyde (w/v) in phosphate-buffered saline (PBS), pH 7.4, for 5 min. Oligonucleotide probes of unique sequence were synthesized on an Applied Biosystem DNA synthesizer. The antisense probe for c-fos (45-mer) was complementary to nucleotides spanning amino acids 1-15 (GCA GCG GGA GGA TGA CGC CTC GTA GTC CGC GTT GAA ACC CGA GAA) [8].The oligonucleotide probe for c-jun (60-mer) was complementary to nucleotides spanning the last 20 amino acids of the predicted protein (GCA ACT GCT GCG TTA GCA TGA GTT GGC ACC CAC TGT TAA CGT GGT TCA TGA CTT TCT GTT) [l]. The antisense probe for Krox-24 was synthesized corresponding t o nucleotides spanning amino acids 2-16 (CCG TTG CTC AGC AGC ATC ATC TCC TCC AGT TTG GGG TAG TTG TCC) [40]. Following the protocol by Wisden et al. [56], probes were 3'-labeled with [alpha-3sS]-dATP (1500 Ci/mmol, New England Nuclear), using terminal Sornmer et al: Selective c-JUN Expression in C A I Neurons transferase from Boehringer (Mannheim, Germany). Radiolabeled probes were diluted 1:100 in hybridization buffer (50% formamide, 4 x saline sodium citrate (SSC), 10% dextran sulphate) and sections were covered by 100 p1 overnight at 42°C. Subsequently, sections were washed as follows: 1 x SSC for 1 h at 56"C, 1 x SSC for 15 min at room temperature (RT), 0.1 x SSC for 15 min at RT. Following dehydration in 7096, 90% and 100% ethanol and air drying, sections were exposed t o Kodak XAR films for 14 days. Control sections were incubated with the labeled antisense probe after prior hybridization with an excess (100-fold) of unlabeled antisense probe [2] which did not result in radioactive labeling (not shown). To standardize exposure times as a control for the saturation range of Kodak XAR films, autoradiographic standards ([*4C]-micro-scales, Amersham, UK), were co-exposed with the sections. Furthermore, sections were repeatedly exposed t o XAR films for different time intervals. Results lmmunocytochernistry Irnrnunocytochemistry. All antibodies were generated in rabbits immunized with bacterially expressed fusion proteins [34, 351. All cDNAs used to construct fusion proteins were of mouse origin. The specificity of the antibodies was previously shown in immunoprecipitation and Western blot experiments [34, 351. To further prove the specificity of the antibodies, preabsorption of the antisera with different antigens was performed [17]. For preabsorption 1 nM and 10 nM fusion protein (in 12.5 mM Tris base, 12.5 mM glycine, 0.01 Yo sodium dodecyl sulfate (SDS), and 30 pm phenylmethylsulfonyl fluoride) were incubated for 24 h with antisera diluted as for immunocytochemistry: c-FOS 1:40.000, FOS B 1:2000, c-JUN 1:1000, JUN B 15000,JUN D 1:lO.OOO and KROX-24 1:lO.OOO. Thereafter, the complex of antiserum and fusion protein was processed for immunocytochemistry. In all cases, immunoreactivity was blocked by 1 nM of the respective antigen. Incubation with 10 nM of other related fusion proteins did not affect immunoreactivity. Immunocytochemistry was performed o n coronal free-floating 50 p m vibratome sections. Sections were incubated in normal swine serum (1OYo in PBS and 0.2% Triton X-100) for 1 h, followed by the primary antisera for 36 h at 4" C. The primary antisera were diluted as described above. Immunoreactivity (IR) was visualized by the avidin biotin complex method (Vectastain, Vector Laboratories, USA), as described previously [l11. Sections were developed in 0.02% diaminobenzidine with 0.02% hydrogen peroxide. The reaction product was intensified by addition of 0.02% cobalt chloride and nickel ammonium sulphate, resulting in black immunostaining. A subset of slides was counterstained with hemalaun. c-JUN. In untreated animals, hippocampal c-JUN IR was confined to the dentate gyrus and CA3 neurons (Fig. 1).As reported previously [28], a single 5 min period of ischemia elicits a transient induction of c-JUN in the dentate gyrus and CA3, which peaks between 3 h and 6 h and subsequently declines to control levels (Fig. 1).CA1 neurons, however, remain immunonegative at all recirculation intervals (Fig. 1). In contrast, prestressing the animals prior to the seco n d 5 min period of ischemia caused a distinct induction of c-JUN IR in CA1 pyramidal cells, with peak levels at 18 h of recirculation (Fig. 1). After 48 h, c-JUN expression in CA1 had returned to baseline (not shown). The initial 2.5 min period of ischemia also elicited c-JUN induction in hippocampal CA1 neurons, with an earlier onset, protracted time-course and a markedly higher intensity of IR compared to the combined ischemia group. c-JUN protein in CA1 started t o accumulate at 3 h of recirculation, peaked at 6 h and persisted up to 24 h (Fig. 1). At 48 h of survival, c-JUN IR was distinctly reduced. The time course of c-JUN expression in all other hippocampal subpopulations closely paralleled that of a single period of 5 min [28] and combined (2.5 min plus 5 min) ischemia (Fig. 1). JUN B, JUN D . Constitutive JUN B expression in untreated and sham-operated control animals was observed in a few scattered hippocampal pyramidal cells. In the combined ischemia group, ischemia elicited strong JUN B induction in dentate gyrus and CA3 neurons with peak levels between 3 h and 6 h. At 48 h after ischemia, JUN B expression was undistinguishable from that of control animals. In CA1, no de novo synthesis of JUN B occurred (not shown). The spatial expression pattern of JUN B induction after 2.5 min initial ischemia largely matched that after combined ischemia, but was more accelerated (not shown). In untreated gerbils, JUN D expression was detected in a few scattered neurons of all hippocampal subpopulations. Compared to JUN B, JUN D induction in both experimental groups, was less pronounced and restricted to CA3 and hilar CA4 neurons with peak levels at 6 h after recirculation. At 18 h, JUN D expression had returned t o baseline levels. Again, CAI neurons remained immunonegative throughout the recirculation period (not shown). c-FOS, FOS B . In untreated and sham-operated animals only a few scattered c-FOS immunoreactive Sommer et al. Selective c-JUN Expression in CA1 Neurons C-JUN 5 min 2.5 min 2.5min 5min Figure 1 Postischemic c-JUN protein expression in the gerbil hippocampus without, during and after tolerance acquisition Data of 5 min ischemia without tolerance induction are included for comparison 1281 In control animals, c-JUN expression is restricted to dentate gyrus and some scattered CA3 neurons In all three experimental groups, distinct c-JUN induction is present in CA3 and dentate gyrus cells at 6 h of recirculation However, only during and after tolerance acquisition hippocampal C A I neurons exhibit a marked c-JUN expression, suggesting a potential role in the mechanisms resulting in neuronal survival (50pm vibratome sections, the scalebar corresponds to 500 pm). neuronal nuclei were present in the hippocampus (Fig. 2). At 3 h of recirculation in the combined ischemia group, moderate induction of c-FOS was found in dentate gyrus, CA3 and hilar CA4 neurons. At 6 h, IR of dentate granule cells was already reduced, and at 12 h after combined ischemia, c-FOS expression had returned to control levels in all hippocampal subfields (Fig. 2). In accordance with in situ hybridization results (see below), CA1 neurons remained immunonegative throughout the recirculation period with the exception of a few rather evenly dispersed nuclei of GABAergic interneurons (Fig. 2). After 2.5 min initial ischemia, the spatiotemporal profile of c-FOS induction was similar, but more accelerated compared to combined ischemia with disappearance of IR already at 3 h after recirculation. Only weak induction of c-FOS was seen in CA3 and CA4 neurons at 3 h. In CA1 pyramidal cells, no cFOS expression was observed at any timepoint investigated (not shown). FOS B IR in control gerbils was restricted to a few scattered nuclei of dentate gyrus cells. Compared to c-FOS, FOS B induction in dentate gyrus and CA3 neurons in the combined ischemia group was slightly delayed and persisted up to 6 h (not shown). Again, after 2.5 min initial ischemia, FOS B induction was more accelerated compared to the combined ischemia group. Similar to c-FOS, CA1 neurons remained immunonegative at all timepoints investigated in both experimental groups (not shown). KROX-24. KROX-24 demonstrated marked constitutive expression in CA1 pyramidal cells (Fig. 2). At 3 h of recirculation after combined ischemia, a distinct induction was observed in dentate granule cells, followed by a marked increase in labeling intensity in CA3 neurons at 6 h. In ischemia-tolerant CA1 neurons, constitutive IR persisted up to 6 h of recirculation, followed by a reduction at 12 h and a secondary increase between 18 h and 24 h after recirculation, finally reaching baseline levels at 48 h (Fig. 2). After a single 2.5 min period of ischemia KROX-24 induction was already seen in dentate granule cells at 1 h, followed by a decline at 3 h, and had virtually disappeared at all subsequent timepoints. In CA3 Sommer et al: Selective c-JUN Expression in CAI Neurons C- FOS KROX - 24 Figure 2 Failure of postischemic induction of c-FOS and KROX-24 protein in CAI neurons in the ischemia-tolerant state. In control animals, c-FOS expression is present only in a few scattered hippocampal neurons and KROX-24 is constitutively expressed in C A I . After combined ischemia (2.5 min + 5 min, 4 days apart) and 6 h of recirculation, c-FoS and KROX-24 induction is restricted to CA3 and CA4. In the dentate gyrus IR has already declined. In contrast t o c-JUN (Fig. 1). no c-FOS or increased KROX-24 protein expression occurs in C A I pyramidal cells. At 18 h, c-FOS IR is indistinguishable from controls in all hippocampal subpopulations. After a transient reduction at 12 h, KROX-24 expression in CAI neurons exhibits a secondary increase between 18 h and 24 h after recirculation and returns to baseline levels at 48 h 150pm vibratome sections; the scalebar corresponds to 500 pm). only weak KROX-24 induction occurred at a single postischemic timepoint (3 h). In CA1 constitutive levels remained unaltered at all timepoints investigated (not shown). In Sifu Hybridization c-jun. Constitutive expression of c-jun mRNA was present in dentate gyrus (DG) and CA3 and just above the level of detection (Fig. 3). At 1 h of recirculation, in particular the DG, but also CA3 neurons in the combined ischemia group exhibited distinct induction of c-jun mRNA with highest labeling intensities in dentate granule cells. At 3 h and 6 h of recovery, labeling had already declined and returned to control at 18 h. In contrast to c-fos (see below), a 5 min period of ischemia after preconditioning caused Sommer et al Selective c-JUN Expression in CAI Neurons c -jun c -fos Krox -24 co Ih 6h 48h Figure 3 Postischemic transcription of c-jun, c-fos and Krox-24 in the gerbil hippocampus after tolerance acquisition (2.5 + 5 min global ischemia 4 days apart). In control gerbils, c-fos mRNA is not detectable, c-jun levels are just above the limits of detection in dentate gyrus (DG) and CA3, and Krox-24 mRNA is constitutively expressed in CAI. During the recirculation period after combined ischemia c-fos hybridization becomes barely detectable at 1 h in DG and disappears thereafter. In contrast, both c-jun and Krox-24 exhibit a rapid (1 h) and transient increase of transcription in DG and CA3. At 6 h, c-jun mRNA hybridization is present in ischemiatolerant CAI neurons. Krox-24 mRNA is also slightly elevated. At 48 h, mRNA expression of all IEGs has returned to baseline levels (negative film images, the scalebar corresponds to 1 mm) a distinct delayed c-jun transcription in CA1 which peaked between 6 h and 12 h of recirculation (Fig. 3). Using a 3%-labeled oligonucleotide, no hybridization signal was detectable in the hippocampus of untreated and sham-operated control gerbils (Fig. 3). At 1 h and 3 h of recirculation, moderate expression of c-fos mRNA in the ischemia tolerant group was restricted to dentate granule cells (1 h) and CA3 neurons (1 h and 3 h) and had returned to baseline at 6 h of recirculation. In contrast to DG and CA3 neurons, no labeling was visible in CA1 pyramic-fos. dal cells at any time point investigated (Fig. 3). Krox-24. In untreated and sham-operated control animals, constitutive expression of Krox-24 mRNA was evident with highest labeling intensity in CA1 (Fig. 3). Gerbils subjected to 5 min ischemia 4 d after prestressing by 2.5 min ischemia, exhibited an induction of Krox-24 transcription in all hippocampal neurons with peak levels in DG at 1 h and in CA3 and CA1 neurons at 3 h, respectively. At 6 h, Krox-24 transcription was already substantially diminished in all hippocampal subpopulations except CA1 (Fig. 3 ) . Sommer et al. Selective c-JUN Expression in CAI Neurons Subsequently mRNA expression declined and at 48 h, Krox-24 had returned to baseline levels (Fig. 3). Discussion The present study investigated the expression profile of cellular IEGs in the gerbil hippocampus during and after acquisition of an ischemia-tolerant state. Protection of CA1 neurons to ischemia was conferred by a priming event, i.e. an initial (2.5 min) nonlethal period of global ischemia [25, 30, 32, 371. ischemia in the tolerant state was performed by a second, normally lethal 5 min period of bilateral carotid occlusion 4 days later. In contrast to 5 min ischemia without tolerance induction [28], the major finding was a selective postischemic induction of only c-JUN in hippocampal CA1 neurons both after preconditioning and after combined ischemia. c-JUN expression did not follow IEG kinetics, but was more protracted during gain of a neuroprotective state (peak at 6 h, persistence up to 24 h of recirculation) and delayed after combined ischemia (peak at 18 h). The results of this study demonstrate that differing ischemia paradigms elicit completely different IEG responses in the adult gerbil CNS. Excitotoxic activation of glutamate receptors by a single 5 min period of transient global ischemia in the gerbil brain is thought to be a key event for the molecular pathogenesis of postischemic delayed neuronal death of CA1 neurons in the hippocampus. Glutamate receptor stimulation is also thought to be a key mediator of postischemic IEG induction. In irreversibly damaged CA1 neurons, however, increased transcription of IEGs is not followed by translation into protein and CA1 neurons remain devoid of newly synthesized transcription factors during the recirculation period [28](Fig. 1). In contrast, early and transient expression of all IEG encoded proteins including c-JUN occur after 5 min ischemia in other hippocampal subpopulations (DG, CA3) that are also targets of glutamate receptor mediated neurotoxicity but destined to survive [28](Fig. 1).This temporospatial distribution pattern with a slightly asynchronous, but rapid postischemic increase (3-6 h) and subsequent decline to baseline levels (<24 h) of all IEG encoded proteins investigated was also observed in dentate granule cells and CA3 neurons in the 2.5 min initial ischemia and the combined ischemia group (Figs. 1 and 2). In contrast t o a single 5 min period of ischemia, however, a n additional and selective induction of c-jun both on the mRNA (combined ischemia group, Fig. 3) and on the protein level (2.5 min initial ischemia and combined ischemia group, Fig. 1) was present in CA1 neurons which remained viable, but are likely t o be severely injured during and despite tolerance acquisition. The presence of JUN-like IR between 6 and 24 h of recirculation in CA1 neurons following 2 min of ischemia has been previously reported by Nowak et al. [44]. Our data corroborate these results and identify c-JUN as the inducible member of the JUN family. Apart from the heat shock/stress response this selective c-JUN induction in ischemia-tolerant CA1 neurons constitutes the only change in gene expression so far detected. The experimental conditions that trigger the selective c-JUN expression in CA1 resemble those of heat shock protein (HSP) 70 induction, which is the hallmark of the heat shock/stress response in rodent brain and appears to be a sensitive indicator of neurons at risk. In analogy to HSP70 expression, c-JUN exhibits a narrow range of inducibility. The threshold of injury for induction of c-JUN and HSP70 expression in CA1 is achieved by 2.5 min initial ischemia and combined ischemia, but is surpassed by the injury inflicted by 5 min ischemia without priming event [SO]. However, compared to c-JUN (peak at 6 h in the 2.5 min initial ischemia group and peak at 18 h in the combined ischemia group, respectively), HSP70 expression (peak at 48 h in both groups) is even more delayed [30]. Heat shock/stress genes in eucaryotes are transcriptionally activated by a pre-existing heat shock factor (HSF)[46]. c-JUN is thus part of a different signalling pathway potentially causing earlier and other molecular alterations associated with tolerance induction. The selective cJUN expression without concomitant induction of other transcription factors must have consequences for the transcriptional activation of target genes. in contrast to FOS proteins, c-JUN can form homodimers and - apart from heterodimerization with the constitutively expressed JUN D - does not need other molecular partners for transcriptional interaction with the AP1 site, although binding occurs with lower affinity [7, 41, 491. A similar finding with selective and prolonged expression of c-JUN protein in hippocampal CA1 neurons and neurons in the immediate vicinity of degenerated dentate granule cells has recently been obtained after severe hypoglycemia in rats (Gass et al., in press). In analogy t o very brief periods of ischemia, hypoglycemia leads to a sublethal but still reversible injury of hippocampal CA1 neurons and granule cells except at t h e crest of t h e dentate gyrus ~41. In contrast to the well-documented persistent deficit of protein synthesis after 5 min ischemia without preconditioning in gerbil CA1 neurons [3, 53, 551, the observed temporal profile of c-JUN protein expression agrees well with data on the recovery of protein synthesis following tolerance induction. Recent studies (10, 421 after combined ischemia demonstrate restoration of impaired amino acid incorporation with reaggregation of ribosomes within 24 h suggesting that tolerance-induced neurons have the capacity to restore protein synthesis during the early postischemic recovery period. There are a variety of experimental protocols in the rat [37] and in the gerbil [25,30, 321 for ischemiatolerance induction with different numbers of and Sommer et al: Selective c-JUN Expression in CA1 Neurons different intervals between the priming events resulting in varying degrees of neuronal protection. The timecourse of the IEG or stress response during and after tolerance acquisition on the translational level may therefore vary with the model of tolerance induction used. Further studies are necessary to clarify this point. Krox-24 is constitutively expressed at relatively high levels in CA1 neurons (Figs. 2 and 3 ) but despite a transient increase of transcription, IR was not unequivocally elevated after combined ischemia (Figs. 2 and 3). In contrast to 5 min ischemia without prestressing which is also associated with a transient upregulation of Krox-24 transcription but followed by an irreversible loss of KROX-24 IR in lethally damaged CA1 neurons [28], 5 min ischemia in the tolerant state caused a reversible decrease of KROX-24 expression between 12 h and 18 h, but at 48 h of recirculation, control levels were re-attained (Fig. 2). Since constitutive KROX-24 expression is thougt to be maintained by NMDA receptor mediated activity [57], the transient decrease of KROX-24 IR in CA1 neurons after combined ischemia may reflect an intermittent decrease of neuronal activity during the recirculation period. A similar transient reduction of KROX-24 levels [12] that correlates with a concomitant decrease in metabolic activity was observed in the neocortex after limbic seizures in rats. Regeneration after axotomy and recovery of neuronal function after ischemia appear t o be unrelated events. Despite fundamental differences in the pathogenetic mechanisms after nerve transsection and ischemia, axotomy results in a block of axoplasmic transport and a retrograde cell body response vs. an excitotoxic, axon-sparing lesion as a consequence of ischemia; recent evidence suggests that they may share some common molecular features. A selective and prolonged expression of c-iun mRNA and c-JUN protein has been described following axotomy or block of axonal transport in peripheral sensory or motor neurons [19, 201. Upregulation of c-JUN expression persisted until complete regeneration. A similar upregulation of c-jun which did not follow IEG kinetics also occurred after axonal damage to central (rubrospinal and nigrostriatal) neurons [21, 221 with a secondary decline in conjunction with the failure of central neurons t o regenerate or persistence up to 150 days [16]. Other authors reported increased c-JUN levels up to one year after grafting striatal neurons to the striatum of adult rats [91. None of these events lead to an upregulatioq of FOS proteins. In fact, it has been suggested that prolonged upregulation of c-JUN, but not c-FOS, is a hallmark of a restorative or regenerative growth response. Even more surprising is the finding that analogous to preconditioning in ischemia, regeneration of spinal cord neurons is greatly facilitated by a preconditioning crush to the peripheral branch of the neuron, which also results in strong expression of c-JUN in damaged neurons 120, 471. Thus, c-JUN induction could be a novel molecular correlate of mechanisms resulting in neuronal regeneration and survival irrespective of the pathogenetic mechanism of cell damage. Acknowledgements The authors gratefully acknowledge the superb technical assistance of Stephan Hennes, Claudia Kammerer, Bettina Brosig and Gillian Muncke. We thank Rodrigo Bravo and Thomas Herdegen for providing antibodies against IEG encoded proteins. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 3 17). Note added in proof After submission of this manuscript, the following paper has come to our attention: Shimazaki K, Ishida A, Kawai N (1994) Increase in bcl-2 oncoprotein and the tolerance to ischemiainduced neuronal death in the gerbil hippocampus. Neurosci Res 20: 95-99 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Brain Pathology Wiley

Selective c‐JUN Expression in CA1 Neurons of the Gerbil Hippocampus during and after Acquisition of an Ischemia‐Tolerant State

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Copyright © 1995 Wiley Subscription Services, Inc., A Wiley Company
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10.1111/j.1750-3639.1995.tb00587.x
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Abstract

Selective c-JUN Expression in C A I Neurons of the Gerbil Hippocampus during and after Acquisition of an Isc hemia-ToIerant State Clemens Sommer, Peter Gass, Marika Kiessling Department of Neuropathology, University of Heidelberg, D-69120 Heidelberg, Germany Introduction The selective delayed neuronal death of CAI pyramidal cells after transient global ischemia i n the gerbil brain can be prevented by preconditioning with a short sublethal period of ischemia 1 - 7 days prior t o a subsequent, usually lethal ischemia of 5 min duration. Since changes of neuronal gene expression may play a crucial role in this tolerance induction, w e investigated the postischemic expression profile of the fos, jun and Krox transcription factor families. We have previously reported that a single 5 min period of cerebral ischemia does not cause a de novo synthesis of immediate early gene (IEG) encoded proteins in CAI neurons. In the present study, t w o experimental groups of Mongolian gerbils were investigated: one group was subjected t o a single tolerance-inducing 2.5 min period of ischemia by bilateral occlusion of the common carotid artery. The second (combined ischemia) group was subjected t o 2.5 m i n of ischemia, followed b y 5 min of ischemia 4 days later. Postischemic expression of c-FOS, FOS B, c-JUN, JUN B, JUN D and KROX-24 was investigated by in situ hybridization and immunocytochemistry up t o 48 h of recirculation. In contrast t o a single 5 min period of ischemia, 2.5 min caused a postischemic expression of c-JUN protein, but no other IEGs, i n C A I neurons (peak at 6 h). Similarly, a selective but delayed c-JUN expression (peak at 18 h) was observed in animals subjected t o combined ischemia. These results indicate that the induction of an endogenous neuroprotective state in CAI neurons is associated with the activation of a genetic program which involves the expression of specific transcription factors. Received 1 1 January 1995 Corresponding author Dr M Kiessling, Institute of Neuropathology, University of Heidelberg, D-69120 Heidelberg, Germany Tel +49 (6221) 56 2603, Fax +49 (6221) 56 3466 Transient global forebrain ischemia of 5 min duration in the gerbil severely affects energy metabolism in several areas of the central nervous system (CNS), but in the hippocampus only CA1 neurons undergo selective and delayed degeneration [29]. Recently, a number of studies have demonstrated that highly vulnerable hippocampal CA1 neurons both in the gerbil [25, 30, 321 and in the rat [37] can be protected significantly against ischemic damage by prestressing with a short sublethal ischemic period. This phenomenon of ischemia-tolerance induction has also been described in other brain regions [14, 311, and after preconditioning by hyperthermic [6, 331 or systemic oxidative stress in vivo [45]. The molecular basis of tolerance induction is not yet understood. Two studies suggested the possibility that loss of hilar somatostatin neurons by short periods of ischemia [39] or destruction of dentate granule cells by colchicine [23] could confer protection of CAI neurons by triggering a modulation of excitatory neurotransmission in the hippocampal circuitry. Considerable interest, however, has been attracted by several investigations which demonstrate a coincidence of tolerance induction and the expression of a 70 kDa heat shock protein (HSP70) in vivo [14, 30, 37, 501, suggesting a possible role of stress proteins in the acquisition of an ischemia-tolerant state. In analogy, in vitro induction of HSP70 in cultured neurons has been shown to be associated with increased tolerance to glutamate toxicity [38, 481. It is well known that living organisms, when exposed t o sublethal environmental stress such as high temperature, amino acid analogues, heavy metals or inhibition of energy metabolism, respond by intrinsic alterations of the molecular phenotype, preferentially by initiating the synthesis of several so called heat shock or stress proteins (for review see [5, 18, 24, 36, 441). However, there is n o direct evidence linking stress protein expression to tolerance induction. Furthermore, recent data indicate that HSP70 induction after global ischemia in the gerbil predominantly depends o n postischemic hyperthermia [52] and in focal ischemia models even occurs after sham occlusion of the middle cerebral artery [14]. Low molecular Sommer et al: Selective c-JUN Expression in C A I Neurons weight stress proteins, e.g. HSP27, also show a temporal expression compatible with the induction of an ischemia-tolerant state, but the distribution pattern makes it unlikely that they are involved in neuroprotection by preconditioning [26]. To the best of our knowledge, no changes in gene expression in CA1 neurons other than the heat shockfstress response have been reported following prestressing. One other gene family potentially involved is the class of immediate early genes (IEGs) which are also known t o respond to a variety of noxious stimuli, including ischemia [ll, 13, 17, 27, 28, 541. IEGs are thought t o be third messengers in a complex cellular cascade of stimulus-transcription coupling that converts extracellular signals into alterations of cellular functions by regulating target gene expression, such as neurotransmitter or neuromodulator genes [15, 43, 511. In Mongolian gerbils subjected to a single 5 min period of transient global ischemia, irreversibly damaged CA1 neurons exhibit strong transcription of various IEGs [28], but probably due to a severe and persistent inhibition of protein synthesis [3, 53, 551, no translation occurs. In contrast, a short priming ischemic period which is sufficient for tolerance aquisition provides the potential for changes in gene expression to become effective. It causes no appreciable neuronal damage, and is associated with recovery of protein synthesis in CA1 neurons [lo, 421. To study the IEG response during tolerance acquisition, we examined the synthesis of six transcription factors of the fos, jun and Krox families in the gerbil hippocampus after a single tolerance-inducing 2.5 min period of ischemia and various recirculation periods. To examine the effect of tolerance acquisition on IEG expression, a second group of gerbils was subjected t o combined 2.5 min and 5 min of forebrain ischemia 4 d apart. For three representative members of different IEG subclasses (c-fos, c-jun, Krox-24) analysis in the combined ischemia group was performed both at the transcriptional and the translational level. Material and Methods (combined ischemia group). Anaesthesia was induced with a mixture of 30% 02, 700/0 N,O, and 1.5% halothane. In both experimental groups the common carotid arteries were exposed and double clamped with atraumatic Biemer aneurysm clips. In both groups clips were removed and restoration of blood flow was visually verified after 2.5 min. In the combined ischemia group animals were resubjected to a 5 min period of ischemia four days later using the same protocol. Expression of six IEG encoded proteins (c-FOS, FOS B, c-JUN, JUN B, JUN D, KROX-24) were investigated immunocytochemically in both experimental groups after recirculation intervals of 3 h, 6 h, 12 h, 18 h, 24 h and 48 h (n=4 per time point). Control gerbils of both groups were subjected to anaesthesia and all surgical procedures, except clamping of the carotid arteries (n=1 per time point). In addition, baseline levels of IEGs were determined in 3 untreated animals to exclude possible effects due to handling and anaesthesia. Animals were sacrificed by transcardiac perfusion under deep ether anaesthesia. Heparin (90 IU) was given via the left ventricle prior to washout of blood vessels with isotonic saline. Brains were perfusion-fixed with 4 % (w/v) paraformaldehyde solution, removed and postfixed overnight in the same fixative prior to vibratome sectioning. Comparative analysis of representative IEG mRNAs (c-fos, c-jun, Krox-24) was carried out in the combined ischemia group by in situ hybridization using the same postischemic time points as for immunocytochemistry plus one additional earlier time point at 1 h of recirculation (n=3 per time point). Animal experiments. Induction of ischemia tolerance was performed according t o the protocol of Kirino et al. [30] with minor modifications. We employed the bilateral carotid occlusion model as already described in detail in previous publications from our laboratory [28, 531. Experiments were performed on adult male Mongolian gerbils (Meriones unguiculatus, 70-80 g) obtained from Tumblebrook Farms (West Brookfield, MA, USA). In addition to the previously studied standard model of a single 5 min period of ischemia [28], two experimental groups of animals were investigated: one group was subjected to a single tolerance inducing 2.5 min period of ischemia (2.5 min initial ischemia group). The second group was subjected to 2.5 min of ischemia, followed by 5 min of ischemia 4 d later In situ hybridization. For in situ hybridization, brains were rapidly removed and frozen in 2-methylbutane at -30°C. Coronal sections 14 pm thick at the level of the dorsal hippocampus were cut in a cryostat at -2O"C, dried o n poly-L-lysine coated slides and fixed in 4% paraformaldehyde (w/v) in phosphate-buffered saline (PBS), pH 7.4, for 5 min. Oligonucleotide probes of unique sequence were synthesized on an Applied Biosystem DNA synthesizer. The antisense probe for c-fos (45-mer) was complementary to nucleotides spanning amino acids 1-15 (GCA GCG GGA GGA TGA CGC CTC GTA GTC CGC GTT GAA ACC CGA GAA) [8].The oligonucleotide probe for c-jun (60-mer) was complementary to nucleotides spanning the last 20 amino acids of the predicted protein (GCA ACT GCT GCG TTA GCA TGA GTT GGC ACC CAC TGT TAA CGT GGT TCA TGA CTT TCT GTT) [l]. The antisense probe for Krox-24 was synthesized corresponding t o nucleotides spanning amino acids 2-16 (CCG TTG CTC AGC AGC ATC ATC TCC TCC AGT TTG GGG TAG TTG TCC) [40]. Following the protocol by Wisden et al. [56], probes were 3'-labeled with [alpha-3sS]-dATP (1500 Ci/mmol, New England Nuclear), using terminal Sornmer et al: Selective c-JUN Expression in C A I Neurons transferase from Boehringer (Mannheim, Germany). Radiolabeled probes were diluted 1:100 in hybridization buffer (50% formamide, 4 x saline sodium citrate (SSC), 10% dextran sulphate) and sections were covered by 100 p1 overnight at 42°C. Subsequently, sections were washed as follows: 1 x SSC for 1 h at 56"C, 1 x SSC for 15 min at room temperature (RT), 0.1 x SSC for 15 min at RT. Following dehydration in 7096, 90% and 100% ethanol and air drying, sections were exposed t o Kodak XAR films for 14 days. Control sections were incubated with the labeled antisense probe after prior hybridization with an excess (100-fold) of unlabeled antisense probe [2] which did not result in radioactive labeling (not shown). To standardize exposure times as a control for the saturation range of Kodak XAR films, autoradiographic standards ([*4C]-micro-scales, Amersham, UK), were co-exposed with the sections. Furthermore, sections were repeatedly exposed t o XAR films for different time intervals. Results lmmunocytochernistry Irnrnunocytochemistry. All antibodies were generated in rabbits immunized with bacterially expressed fusion proteins [34, 351. All cDNAs used to construct fusion proteins were of mouse origin. The specificity of the antibodies was previously shown in immunoprecipitation and Western blot experiments [34, 351. To further prove the specificity of the antibodies, preabsorption of the antisera with different antigens was performed [17]. For preabsorption 1 nM and 10 nM fusion protein (in 12.5 mM Tris base, 12.5 mM glycine, 0.01 Yo sodium dodecyl sulfate (SDS), and 30 pm phenylmethylsulfonyl fluoride) were incubated for 24 h with antisera diluted as for immunocytochemistry: c-FOS 1:40.000, FOS B 1:2000, c-JUN 1:1000, JUN B 15000,JUN D 1:lO.OOO and KROX-24 1:lO.OOO. Thereafter, the complex of antiserum and fusion protein was processed for immunocytochemistry. In all cases, immunoreactivity was blocked by 1 nM of the respective antigen. Incubation with 10 nM of other related fusion proteins did not affect immunoreactivity. Immunocytochemistry was performed o n coronal free-floating 50 p m vibratome sections. Sections were incubated in normal swine serum (1OYo in PBS and 0.2% Triton X-100) for 1 h, followed by the primary antisera for 36 h at 4" C. The primary antisera were diluted as described above. Immunoreactivity (IR) was visualized by the avidin biotin complex method (Vectastain, Vector Laboratories, USA), as described previously [l11. Sections were developed in 0.02% diaminobenzidine with 0.02% hydrogen peroxide. The reaction product was intensified by addition of 0.02% cobalt chloride and nickel ammonium sulphate, resulting in black immunostaining. A subset of slides was counterstained with hemalaun. c-JUN. In untreated animals, hippocampal c-JUN IR was confined to the dentate gyrus and CA3 neurons (Fig. 1).As reported previously [28], a single 5 min period of ischemia elicits a transient induction of c-JUN in the dentate gyrus and CA3, which peaks between 3 h and 6 h and subsequently declines to control levels (Fig. 1).CA1 neurons, however, remain immunonegative at all recirculation intervals (Fig. 1). In contrast, prestressing the animals prior to the seco n d 5 min period of ischemia caused a distinct induction of c-JUN IR in CA1 pyramidal cells, with peak levels at 18 h of recirculation (Fig. 1). After 48 h, c-JUN expression in CA1 had returned to baseline (not shown). The initial 2.5 min period of ischemia also elicited c-JUN induction in hippocampal CA1 neurons, with an earlier onset, protracted time-course and a markedly higher intensity of IR compared to the combined ischemia group. c-JUN protein in CA1 started t o accumulate at 3 h of recirculation, peaked at 6 h and persisted up to 24 h (Fig. 1). At 48 h of survival, c-JUN IR was distinctly reduced. The time course of c-JUN expression in all other hippocampal subpopulations closely paralleled that of a single period of 5 min [28] and combined (2.5 min plus 5 min) ischemia (Fig. 1). JUN B, JUN D . Constitutive JUN B expression in untreated and sham-operated control animals was observed in a few scattered hippocampal pyramidal cells. In the combined ischemia group, ischemia elicited strong JUN B induction in dentate gyrus and CA3 neurons with peak levels between 3 h and 6 h. At 48 h after ischemia, JUN B expression was undistinguishable from that of control animals. In CA1, no de novo synthesis of JUN B occurred (not shown). The spatial expression pattern of JUN B induction after 2.5 min initial ischemia largely matched that after combined ischemia, but was more accelerated (not shown). In untreated gerbils, JUN D expression was detected in a few scattered neurons of all hippocampal subpopulations. Compared to JUN B, JUN D induction in both experimental groups, was less pronounced and restricted to CA3 and hilar CA4 neurons with peak levels at 6 h after recirculation. At 18 h, JUN D expression had returned t o baseline levels. Again, CAI neurons remained immunonegative throughout the recirculation period (not shown). c-FOS, FOS B . In untreated and sham-operated animals only a few scattered c-FOS immunoreactive Sommer et al. Selective c-JUN Expression in CA1 Neurons C-JUN 5 min 2.5 min 2.5min 5min Figure 1 Postischemic c-JUN protein expression in the gerbil hippocampus without, during and after tolerance acquisition Data of 5 min ischemia without tolerance induction are included for comparison 1281 In control animals, c-JUN expression is restricted to dentate gyrus and some scattered CA3 neurons In all three experimental groups, distinct c-JUN induction is present in CA3 and dentate gyrus cells at 6 h of recirculation However, only during and after tolerance acquisition hippocampal C A I neurons exhibit a marked c-JUN expression, suggesting a potential role in the mechanisms resulting in neuronal survival (50pm vibratome sections, the scalebar corresponds to 500 pm). neuronal nuclei were present in the hippocampus (Fig. 2). At 3 h of recirculation in the combined ischemia group, moderate induction of c-FOS was found in dentate gyrus, CA3 and hilar CA4 neurons. At 6 h, IR of dentate granule cells was already reduced, and at 12 h after combined ischemia, c-FOS expression had returned to control levels in all hippocampal subfields (Fig. 2). In accordance with in situ hybridization results (see below), CA1 neurons remained immunonegative throughout the recirculation period with the exception of a few rather evenly dispersed nuclei of GABAergic interneurons (Fig. 2). After 2.5 min initial ischemia, the spatiotemporal profile of c-FOS induction was similar, but more accelerated compared to combined ischemia with disappearance of IR already at 3 h after recirculation. Only weak induction of c-FOS was seen in CA3 and CA4 neurons at 3 h. In CA1 pyramidal cells, no cFOS expression was observed at any timepoint investigated (not shown). FOS B IR in control gerbils was restricted to a few scattered nuclei of dentate gyrus cells. Compared to c-FOS, FOS B induction in dentate gyrus and CA3 neurons in the combined ischemia group was slightly delayed and persisted up to 6 h (not shown). Again, after 2.5 min initial ischemia, FOS B induction was more accelerated compared to the combined ischemia group. Similar to c-FOS, CA1 neurons remained immunonegative at all timepoints investigated in both experimental groups (not shown). KROX-24. KROX-24 demonstrated marked constitutive expression in CA1 pyramidal cells (Fig. 2). At 3 h of recirculation after combined ischemia, a distinct induction was observed in dentate granule cells, followed by a marked increase in labeling intensity in CA3 neurons at 6 h. In ischemia-tolerant CA1 neurons, constitutive IR persisted up to 6 h of recirculation, followed by a reduction at 12 h and a secondary increase between 18 h and 24 h after recirculation, finally reaching baseline levels at 48 h (Fig. 2). After a single 2.5 min period of ischemia KROX-24 induction was already seen in dentate granule cells at 1 h, followed by a decline at 3 h, and had virtually disappeared at all subsequent timepoints. In CA3 Sommer et al: Selective c-JUN Expression in CAI Neurons C- FOS KROX - 24 Figure 2 Failure of postischemic induction of c-FOS and KROX-24 protein in CAI neurons in the ischemia-tolerant state. In control animals, c-FOS expression is present only in a few scattered hippocampal neurons and KROX-24 is constitutively expressed in C A I . After combined ischemia (2.5 min + 5 min, 4 days apart) and 6 h of recirculation, c-FoS and KROX-24 induction is restricted to CA3 and CA4. In the dentate gyrus IR has already declined. In contrast t o c-JUN (Fig. 1). no c-FOS or increased KROX-24 protein expression occurs in C A I pyramidal cells. At 18 h, c-FOS IR is indistinguishable from controls in all hippocampal subpopulations. After a transient reduction at 12 h, KROX-24 expression in CAI neurons exhibits a secondary increase between 18 h and 24 h after recirculation and returns to baseline levels at 48 h 150pm vibratome sections; the scalebar corresponds to 500 pm). only weak KROX-24 induction occurred at a single postischemic timepoint (3 h). In CA1 constitutive levels remained unaltered at all timepoints investigated (not shown). In Sifu Hybridization c-jun. Constitutive expression of c-jun mRNA was present in dentate gyrus (DG) and CA3 and just above the level of detection (Fig. 3). At 1 h of recirculation, in particular the DG, but also CA3 neurons in the combined ischemia group exhibited distinct induction of c-jun mRNA with highest labeling intensities in dentate granule cells. At 3 h and 6 h of recovery, labeling had already declined and returned to control at 18 h. In contrast to c-fos (see below), a 5 min period of ischemia after preconditioning caused Sommer et al Selective c-JUN Expression in CAI Neurons c -jun c -fos Krox -24 co Ih 6h 48h Figure 3 Postischemic transcription of c-jun, c-fos and Krox-24 in the gerbil hippocampus after tolerance acquisition (2.5 + 5 min global ischemia 4 days apart). In control gerbils, c-fos mRNA is not detectable, c-jun levels are just above the limits of detection in dentate gyrus (DG) and CA3, and Krox-24 mRNA is constitutively expressed in CAI. During the recirculation period after combined ischemia c-fos hybridization becomes barely detectable at 1 h in DG and disappears thereafter. In contrast, both c-jun and Krox-24 exhibit a rapid (1 h) and transient increase of transcription in DG and CA3. At 6 h, c-jun mRNA hybridization is present in ischemiatolerant CAI neurons. Krox-24 mRNA is also slightly elevated. At 48 h, mRNA expression of all IEGs has returned to baseline levels (negative film images, the scalebar corresponds to 1 mm) a distinct delayed c-jun transcription in CA1 which peaked between 6 h and 12 h of recirculation (Fig. 3). Using a 3%-labeled oligonucleotide, no hybridization signal was detectable in the hippocampus of untreated and sham-operated control gerbils (Fig. 3). At 1 h and 3 h of recirculation, moderate expression of c-fos mRNA in the ischemia tolerant group was restricted to dentate granule cells (1 h) and CA3 neurons (1 h and 3 h) and had returned to baseline at 6 h of recirculation. In contrast to DG and CA3 neurons, no labeling was visible in CA1 pyramic-fos. dal cells at any time point investigated (Fig. 3). Krox-24. In untreated and sham-operated control animals, constitutive expression of Krox-24 mRNA was evident with highest labeling intensity in CA1 (Fig. 3). Gerbils subjected to 5 min ischemia 4 d after prestressing by 2.5 min ischemia, exhibited an induction of Krox-24 transcription in all hippocampal neurons with peak levels in DG at 1 h and in CA3 and CA1 neurons at 3 h, respectively. At 6 h, Krox-24 transcription was already substantially diminished in all hippocampal subpopulations except CA1 (Fig. 3 ) . Sommer et al. Selective c-JUN Expression in CAI Neurons Subsequently mRNA expression declined and at 48 h, Krox-24 had returned to baseline levels (Fig. 3). Discussion The present study investigated the expression profile of cellular IEGs in the gerbil hippocampus during and after acquisition of an ischemia-tolerant state. Protection of CA1 neurons to ischemia was conferred by a priming event, i.e. an initial (2.5 min) nonlethal period of global ischemia [25, 30, 32, 371. ischemia in the tolerant state was performed by a second, normally lethal 5 min period of bilateral carotid occlusion 4 days later. In contrast to 5 min ischemia without tolerance induction [28], the major finding was a selective postischemic induction of only c-JUN in hippocampal CA1 neurons both after preconditioning and after combined ischemia. c-JUN expression did not follow IEG kinetics, but was more protracted during gain of a neuroprotective state (peak at 6 h, persistence up to 24 h of recirculation) and delayed after combined ischemia (peak at 18 h). The results of this study demonstrate that differing ischemia paradigms elicit completely different IEG responses in the adult gerbil CNS. Excitotoxic activation of glutamate receptors by a single 5 min period of transient global ischemia in the gerbil brain is thought to be a key event for the molecular pathogenesis of postischemic delayed neuronal death of CA1 neurons in the hippocampus. Glutamate receptor stimulation is also thought to be a key mediator of postischemic IEG induction. In irreversibly damaged CA1 neurons, however, increased transcription of IEGs is not followed by translation into protein and CA1 neurons remain devoid of newly synthesized transcription factors during the recirculation period [28](Fig. 1). In contrast, early and transient expression of all IEG encoded proteins including c-JUN occur after 5 min ischemia in other hippocampal subpopulations (DG, CA3) that are also targets of glutamate receptor mediated neurotoxicity but destined to survive [28](Fig. 1).This temporospatial distribution pattern with a slightly asynchronous, but rapid postischemic increase (3-6 h) and subsequent decline to baseline levels (<24 h) of all IEG encoded proteins investigated was also observed in dentate granule cells and CA3 neurons in the 2.5 min initial ischemia and the combined ischemia group (Figs. 1 and 2). In contrast t o a single 5 min period of ischemia, however, a n additional and selective induction of c-jun both on the mRNA (combined ischemia group, Fig. 3) and on the protein level (2.5 min initial ischemia and combined ischemia group, Fig. 1) was present in CA1 neurons which remained viable, but are likely t o be severely injured during and despite tolerance acquisition. The presence of JUN-like IR between 6 and 24 h of recirculation in CA1 neurons following 2 min of ischemia has been previously reported by Nowak et al. [44]. Our data corroborate these results and identify c-JUN as the inducible member of the JUN family. Apart from the heat shock/stress response this selective c-JUN induction in ischemia-tolerant CA1 neurons constitutes the only change in gene expression so far detected. The experimental conditions that trigger the selective c-JUN expression in CA1 resemble those of heat shock protein (HSP) 70 induction, which is the hallmark of the heat shock/stress response in rodent brain and appears to be a sensitive indicator of neurons at risk. In analogy to HSP70 expression, c-JUN exhibits a narrow range of inducibility. The threshold of injury for induction of c-JUN and HSP70 expression in CA1 is achieved by 2.5 min initial ischemia and combined ischemia, but is surpassed by the injury inflicted by 5 min ischemia without priming event [SO]. However, compared to c-JUN (peak at 6 h in the 2.5 min initial ischemia group and peak at 18 h in the combined ischemia group, respectively), HSP70 expression (peak at 48 h in both groups) is even more delayed [30]. Heat shock/stress genes in eucaryotes are transcriptionally activated by a pre-existing heat shock factor (HSF)[46]. c-JUN is thus part of a different signalling pathway potentially causing earlier and other molecular alterations associated with tolerance induction. The selective cJUN expression without concomitant induction of other transcription factors must have consequences for the transcriptional activation of target genes. in contrast to FOS proteins, c-JUN can form homodimers and - apart from heterodimerization with the constitutively expressed JUN D - does not need other molecular partners for transcriptional interaction with the AP1 site, although binding occurs with lower affinity [7, 41, 491. A similar finding with selective and prolonged expression of c-JUN protein in hippocampal CA1 neurons and neurons in the immediate vicinity of degenerated dentate granule cells has recently been obtained after severe hypoglycemia in rats (Gass et al., in press). In analogy t o very brief periods of ischemia, hypoglycemia leads to a sublethal but still reversible injury of hippocampal CA1 neurons and granule cells except at t h e crest of t h e dentate gyrus ~41. In contrast to the well-documented persistent deficit of protein synthesis after 5 min ischemia without preconditioning in gerbil CA1 neurons [3, 53, 551, the observed temporal profile of c-JUN protein expression agrees well with data on the recovery of protein synthesis following tolerance induction. Recent studies (10, 421 after combined ischemia demonstrate restoration of impaired amino acid incorporation with reaggregation of ribosomes within 24 h suggesting that tolerance-induced neurons have the capacity to restore protein synthesis during the early postischemic recovery period. There are a variety of experimental protocols in the rat [37] and in the gerbil [25,30, 321 for ischemiatolerance induction with different numbers of and Sommer et al: Selective c-JUN Expression in CA1 Neurons different intervals between the priming events resulting in varying degrees of neuronal protection. The timecourse of the IEG or stress response during and after tolerance acquisition on the translational level may therefore vary with the model of tolerance induction used. Further studies are necessary to clarify this point. Krox-24 is constitutively expressed at relatively high levels in CA1 neurons (Figs. 2 and 3 ) but despite a transient increase of transcription, IR was not unequivocally elevated after combined ischemia (Figs. 2 and 3). In contrast to 5 min ischemia without prestressing which is also associated with a transient upregulation of Krox-24 transcription but followed by an irreversible loss of KROX-24 IR in lethally damaged CA1 neurons [28], 5 min ischemia in the tolerant state caused a reversible decrease of KROX-24 expression between 12 h and 18 h, but at 48 h of recirculation, control levels were re-attained (Fig. 2). Since constitutive KROX-24 expression is thougt to be maintained by NMDA receptor mediated activity [57], the transient decrease of KROX-24 IR in CA1 neurons after combined ischemia may reflect an intermittent decrease of neuronal activity during the recirculation period. A similar transient reduction of KROX-24 levels [12] that correlates with a concomitant decrease in metabolic activity was observed in the neocortex after limbic seizures in rats. Regeneration after axotomy and recovery of neuronal function after ischemia appear t o be unrelated events. Despite fundamental differences in the pathogenetic mechanisms after nerve transsection and ischemia, axotomy results in a block of axoplasmic transport and a retrograde cell body response vs. an excitotoxic, axon-sparing lesion as a consequence of ischemia; recent evidence suggests that they may share some common molecular features. A selective and prolonged expression of c-iun mRNA and c-JUN protein has been described following axotomy or block of axonal transport in peripheral sensory or motor neurons [19, 201. Upregulation of c-JUN expression persisted until complete regeneration. A similar upregulation of c-jun which did not follow IEG kinetics also occurred after axonal damage to central (rubrospinal and nigrostriatal) neurons [21, 221 with a secondary decline in conjunction with the failure of central neurons t o regenerate or persistence up to 150 days [16]. Other authors reported increased c-JUN levels up to one year after grafting striatal neurons to the striatum of adult rats [91. None of these events lead to an upregulatioq of FOS proteins. In fact, it has been suggested that prolonged upregulation of c-JUN, but not c-FOS, is a hallmark of a restorative or regenerative growth response. Even more surprising is the finding that analogous to preconditioning in ischemia, regeneration of spinal cord neurons is greatly facilitated by a preconditioning crush to the peripheral branch of the neuron, which also results in strong expression of c-JUN in damaged neurons 120, 471. Thus, c-JUN induction could be a novel molecular correlate of mechanisms resulting in neuronal regeneration and survival irrespective of the pathogenetic mechanism of cell damage. Acknowledgements The authors gratefully acknowledge the superb technical assistance of Stephan Hennes, Claudia Kammerer, Bettina Brosig and Gillian Muncke. We thank Rodrigo Bravo and Thomas Herdegen for providing antibodies against IEG encoded proteins. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 3 17). Note added in proof After submission of this manuscript, the following paper has come to our attention: Shimazaki K, Ishida A, Kawai N (1994) Increase in bcl-2 oncoprotein and the tolerance to ischemiainduced neuronal death in the gerbil hippocampus. Neurosci Res 20: 95-99

Journal

Brain Pathology – Wiley

Published: Apr 1, 1995

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