Delayed and differential induction of p38 MAPK isoforms in microglia and astrocytes in the brain after transient global ischemia

Molecular Brain Research 107 (2002) 137–144 www.elsevier.com / locate / molbrainres

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Delayed and differential induction of p38 MAPK isoforms in microglia and astrocytes in the brain after transient global ischemia Chun Shu Piao a , Yongzhe Che a , Pyung-Lim Han b , Ja-Kyeong Lee a , * a

Department of Anatomy, Inha University School of Medicine, 7 -241 Shinheung-dong, Jung-Gu, Inchon 400 -712, South Korea b Department of Neuroscience and EIN, Ewha Womans University School of Medicine, Seoul, South Korea Accepted 17 July 2002

Abstract The p38 MAPK signaling pathway has been implicated in various pathological conditions of neuronal and non-neuronal cells. Here we report the differential induction of p38 MAPK isoforms, p38a and p38b, in the adult gerbil brain following transient global ischemia. The p38a and p38b kinase activities were gradually enhanced with the peak activity occurring around 2–4 days after ischemic insult. Immunohistochemical analysis revealed that p38a expression was increased as early as 4 h after ischemic insult and enhanced further reaching maximum induction around 4 days after ischemia. The induced p38a was concentrated in microglia in hippocampus as well as in frontal and parietal cortices of the brain, where significant neuronal damage was occurred. By contrast, immunostaining with anti-p38b antibody indicated that p38b was markedly induced in astrocytes in hippocampus around 4 days after ischemic insult, which lasted for the next several days. The differential induction of p38 MAPK isoforms following transient global ischemia, especially the induction of p38a and p38b MAPKs in microglia and astrocytes, respectively, in different time points after ischemic insult suggest distinct roles of p38 MAPK isoforms in post-ischemic brain.  2002 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Ischemia Keywords: p38 MAPK; Global ischemia; Microglia; Astrocytes

1. Introduction The p38 kinase, a member of the mitogen-activated protein (MAP) kinase, is activated by LPS, proinflammatory cytokines, and various environmental stresses including osmotic shock [9]. The activation of p38 MAPK requires phosphorylation of Thr180 and Tyr182 residues within a TGY motif [25], which is carried out by dualspecificity MAPK kinase (MKKs), MKK3 and MKK6 [5]. Upon activation, p38 MAPK phosphorylates various subAbbreviations: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MKK, MAP kinase kinase; MAPKAPK2, MAPK-activated protein kinase 2; ATF2, activating transcription factor; GST, glutathione S-transferase; GFAP, glial fibrillary acidic protein; LPS, lipopolysaccharide *Corresponding author. Tel.: 182-32-890-0913; fax: 182-32-8842105. E-mail address: [email protected] (J.K. Lee).

strates including MAPK-activated protein kinase 2 (MAPKAPK2) [20] and several transcription factors including activating transcription factor 2 (ATF2) [25]. The activation of p38 MAPKs has been implicated in pathological changes in inflammatory and apoptotic processes [9]. Crucial roles of p38 MAPK have been reported in neuronal cell death induced by excitotoxicity or growth factor deprivation in cultured cells [13,15,31]. Recently, several in vivo studies revealed that p38 MAPK is activated in the human brain with Alzheimer’s disease [8] and in the brain of rat or gerbil following ischemia [22,29]. In addition, Sugino et al. [27] reported that the pretreatment of SB203580, an inhibitor of p38 MAPK, reduced the ischemic neuronal death in the CA1 region after transient global ischemia. In this work, we investigated activation and expression of the two p38 MAPK isoforms, p38a and p38b, in the brain following transient global ischemia. Previously, we

0169-328X / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 02 )00456-4

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demonstrated that two p38 MAPK isoforms, p38a and p38b, were abundantly, but differentially, expressed in the brain [18]. Here, we report the differential induction of p38 MAPKs in microglia and astrocytes in adult gerbil brain at different time points after transient global ischemia. These results suggest that two p38 MAPK isoforms, p38a and p38b, may play distinct roles in the post-ischemic brain.

2. Materials and methods

2.1. Materials Adult male gerbils (60–70 g) were housed under 12 h light–dark cycle with free access to food and water. Polyclonal anti-p38a and anti-p38b antibodies were obtained from Santa Cruz (Santa Cruz, CA) and anti-GFAP antibody was from Chemicon (Temecula, CA). HRPconjugated isolectin B4 from Griffonia simplicifolia seeds (GSA I-B4) and FITC-conjugated GSA I-B4 were purchased from Sigma (St. Louis, MO). Rhodamine conjugated anti-rabbit IgG was from Jackson ImmunoRes Lab (West Grove, PA).

2.2. Global ischemia All experiments were carried out in accordance with the guidelines for animal research at Inha University School of Medicine. Surgical procedures for bilateral common carotid artery occlusion were performed as described [14]. Adult male Mongolian gerbils, weighing 60–70 g, were anesthetized with ketamine (50 mg / kg) and xylazine hydrochloride (2.5 mg / kg). Bilateral common carotid arteries were dissected and occluded with microaneurysmal clips for 10 min. During the operation and until they were awake, body temperature was monitored by the rectal thermometer, and maintained at 37 8C. In sham-operated group, the same procedure was performed without carotid occlusion.

2.3. Western analysis Western analyses with hippocampus homogenates were described previously [16]. Briefly, brain was quickly removed from gerbil sacrificed by cervical dislocation, and hippocampus was isolated and placed in ice-cold homogenization buffer (10 mM Tris–HCl, pH 7.4, 0.5 mM EDTA, 0.25 M sucrose and 2 mM PMSF) for homogenization. Each lane in Western blot contains 30 mg of total protein, which was resolved on 12% sodium dodecyl sulfate (SDS)–polyacrylamide gel and subjected to Western blot analysis. Primary antibodies were diluted 1:1000 for antip38a and anti-GFAP and 1:750 for anti-p38b. Signals were visualized by using ECL kit according to the manufacturer’s instructions (Amersham, UK).

2.4. Immunoprecipitation and kinase assays Immunoprecipitation and following kinase assays were described previously [16]. Briefly, hippocampus was homogenized in 50 mM Tris–HCl, pH 7.5 buffer containing 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1% Nonidet P-40, 0.5% deoxycholate, and 0.1% sodium dodecyl sulfate. Approximately 1 mg of proteins was incubated with each p38 isoform-specific antibody for 1 h at 4 8C, and precipitated. Immunopellets were incubated for 30 min at 30 8C with 2 mCi [g- 32 P]ATP, 3 mg substrate proteins in 20 ml of kinase reaction buffer (20 mM HEPES, pH 7.5, 1 mM MgCl 2 , 2 mM DTT, 0.2 mM sodium vanadate) as described previously [16]. GST-ATF2 was used as a substrate for p38 kinase assay.

2.5. Immunohistochemistry Brain sections were prepared as described [17]. Briefly, animals were anaesthetized with 10% chloral hydrate (400 mg / kg, i.p.). The perfusion was carried out with 50 ml of saline through a cannula placed into the left ventricle, which was followed by fixation with 50–60 ml of 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS) kept at 4 8C. Brain was quickly removed, fixed with 4% paraformaldehyde overnight at 4 8C and kept until use for up to 1 week. Immunohistochemistry using floating method was carried out as described [17]. Briefly, brains were cut at 30 mm with vibratome, and sections were incubated in blocking solution containing 5% normal goat serum and 2% BSA in PBS, and then with each primary antibody for 16–18 h at 4 8C. Primary antibodies were diluted 1:500 for anti-p38a, anti-GFAP and 1:200 for anti-p38b. Signals were visualized by the HRP/ DAB system (Vector, Burlingame, CA). For double immunostaining with p38a and GSA-IB4, rhodamine-labeled antigoat IgG (final dilution 1:100) was used as the secondary antibody for polyclonal anti-p38a antibody. After washing with PBS containing 0.1% Triton X-100, sections were incubated with fluorescein isothiocyanate-conjugated GSA I-B4 in PBS for 30 min at room temperature. For double immunostaining with p38b, sections were incubated overnight at 4 8C with a mixture of polyclonal anti-p38b antibody and monoclonal anti-GFAP antibody. Then, a mixture containing fluorescein isothiocyanate-labeled antimouse IgG (final dilution 1:100) and rhodamine-labeled anti-goat IgG (final dilution 1:100) was incubated for 30 min at room temperature.

2.6. Lectin histochemistry Vibratome sections were placed for lectin labeling as described by Streit (1990) [26]. Briefly, the sections were incubated with the Griffonia simplicifolia B4-isolectinHRP conjugate at 10 mg / ml in PBS containing divalent cations either at room temperature for 2–3 h or at 4 8C

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overnight. After three washes in PBS and the localization of lectin binding sites was visualized by using DAB-H 2 O 2 as a peroxidase substrate. The sections were then mounted onto gelatinized slides, dehydrated, cleared in xylene, and mounted.

3. Results

3.1. Activation of p38a and p38b MAPKs after transient global ischemia Towards understanding the in vivo role of p38 MAPKs in neuropathological brain, we were interested in exploring the expression and activity of p38 MAPKs in the gerbil brain insulted by transient global ischemia. The kinase activity of p38 MAPKs was measured using isoformspecific p38 antibodies with focus on the hippocampus at different time points after transient global ischemic insult. The specificity of antibodies was tested by the crossreactivity between antibodies with anti-p38a or anti-p38b immunoprecipitants prepared from gerbil brain homogenates. The result revealed that polyclonal anti-p38a antibody detected 38 kDa band from anti-p38a immunoprecipitant, but not from anti-p38b immuno-precipitant. Similarly, polyclonal anti-p38b antibody detected approximately 40 kDa band from anti-p38b immuno-pellet, which may be comparable to the size of p38b detected in mice [18] (data not shown). We observed that overall the kinase activities of both p38a and p38b were significantly enhanced in the hippocampus 2–4 days after ischemia (Fig. 1). The p38a kinase activity was increased approximately 2-fold 1 day after ischemia (Fig. 1A). Two days after ischemic insult, the p38a activity reached the peak level (Fig. 1A). After that, it decreased and went back to the basal level when examined 7 days after ischemia (data not shown). While the p38b showed a delayed and gradual increase of its activity, the p38b activity began to increase 2 days after ischemic insult and reached the 2.9-fold of induction 4 days after the insult (Fig. 1B). This enhanced activity of p38b was maintained for the next few more days (data not shown). In contrast to the enhanced kinase activity, the total amounts of p38a or p38b levels in the hippocampus were unchanged during this period as determined by Western blot anlyses (Fig. 1).

3.2. Induction of p38a in microglia in hippocampus following transient global ischemia To determine the cellular localization of p38 MAPK isoforms, we performed immunohistochemistry using p38 MAPK isoform-specific antibodies. Previously, we reported the constitutive expression and high basal activity of p38a in the adult brain. In hippocampus of normal brain, p38a was expressed mainly in neurons in the pyramidal layer as well as in the dentate gyrus [18]. In

Fig. 1. Activation of p38 MAPKs in hippocampus after transient global ischemia. Kinase activity and protein levels of p38a (A) and p38b (B) in hippocampus were presented at different time points after transient global ischemia. The kinase activities for both p38a and p38b in hippocampus were notably enhanced in the post-ischemic brain, but with a slightly different time frame. Results were represented as the average6S.E.M. of fold induction from three separate experiments. In Western blot analysis, anti-p38a and anti-p38b antibodies recognized, respectively, 38 and 40 kDa bands of proteins. No significant change in the amount of proteins was detected. Bars, amount of protein: solid bars, kinase activity.

sham-operated animals, in addition to the basal expression ¨ animals, weak induction of p38a similar to that in naıve immunoreactivity was detected in cells located outside the pyramidal cell layer of hippocampus (Fig. 2A and B). Moderate induction of p38a immunoreactivity began to occur as early as 4 h after ischemic insult (data not shown). Twenty-four hours after ischemia, the p38a immunoreactivity became augmented in all areas of hippocampus (Fig. 2C and D) and continued to increase further. Four days after ischemia, significant induction of p38a was detected in the hippocampus with the prominent enrichment in the stratum oriens and in pyramidal layer of CA1 region (Fig. 2E and F). It may be worth noting that within pyramidal cell layer, the p38a-positive cells were concentrated in the CA1 region of hippocampus (Fig. 2E), where severe neuronal death was undergoing as a result of transient global ischemia. The p38a-positive cells were

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Fig. 2. Immunohistochemical localization of p38a in hippocampus after transient global ischemia. Brain sections obtained from animals with sham-operated (A, B) or 1 day (C, D) or 4 days (E, F) after 10 min transient global ischemia were immunostained with anti-p38a antibody. The induction of p38a-immunoreactivity began to be detected 1 day after ischemic insult (C, D) and continued to increase until 4 days after ischemic insult (E, F). The p38a-immunoreactive cells resembled microglia, in which p38a-immunoreactivity was localized mainly in their nucleus (F, inset). Scale bars represent 300 mm for A and 50 mm for B.

small with an elongated cell body, and had one or more thick processes with numerous short secondary branches along their length (Fig. 2F, inset), showing characteristics of activated microglia. The p38a expression in microglia declined beginning 4 days after ischemia.

3.3. Delayed induction of p38b in astrocytes in hippocampus following transient global ischemia Compared to p38a, the induction of p38b started to occur at a later time point. The induction of p38b expression appeared 3–4 days after ischemic insult. In

sham-operated animals, the baseline immunoreactivity was detected in the pyramidal cell layer of whole hippocampus and in the dentate gyrus (Fig. 3A and B), which was similar to those described previously [18]. Three days after ischemic insult, the induction of p38b immunoreactivity appeared in the cells located outside the pyramidal cell layer in the CA1 region of hippocampus (data not shown). The p38b expression was further increased at 4 days after ischemia, when the p38b staining was extended to the whole hippocampus (Fig. 3C and D). The p38b-immunoreactive cells seemed to be reactive astrocytes (Fig. 3D, inset), since they resembled anti-GFAP-positive astrocytes detected in adjacent sections (data not shown). In contrast

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Fig. 3. Immunohistochemical localization of p38b in hippocampus after transient global ischemia. Brain sections obtained from animals with sham-operated (A, B) or 4 days (C, D) after 10 min transient global ischemia were immunostained with anti-p38b antibody. The significant induction of p38b-immunoreactivity was detected in scattered cells in the whole hippocampus (C). The morphology of anti-p38b-immunoreactive cells resembled reactive astrocytes, in which the immunoreactivity of p38b was concentrated in the nucleus of cells (D, inset). Scale bars represent 300 mm for A and 50 mm for B.

to the expression of p38a, the induced p38b expression in astrocytes lasted for the next several days (data not shown).

3.4. p38a and p38b were expressed in microglia and astrocytes, respectively Double immunofluorescent staining was employed to determine the identity of the p38a- or p38b-positive cells. The immunostaining was carried out with hippocampal sections prepared 4 days after transient global ischemia. Double staining with anti-p38a (Fig. 4A) and Griffonia simplicifolia isolectin-B4 (GSA I-B4), a microglial marker (Fig. 4B), showed that p38a was expressed in the GSA I-B4-positive cells located in the striatum radiatum in the CA1 region (Fig. 4C). On the contrary, double immunostaining with anti-p38b (Fig. 4D) and anti-GFAP (Fig. 4E) antibodies revealed that p38b was localized in GFAPpositive cells located in the hippocampus, demonstrating that the p38b-positive cells were astrocytes (Fig. 4F). Very interestingly, the induction of p38a and p38b were concentrated in the nucleus of microglia and astrocytes, respectively.

3.5. Induction of p38 MAPK in neocortex after global ischemia The induction of p38a was also observed in the cerebral

cortex, where the overall level of the induction was lower than that in hippocampus. The p38a induction began to occur around 1 day after ischemia, when the immunoreactivity was detected more strongly in small-sized cells scattered in layers 2 and 3 (Fig. 5B). Four days after ischemia, the number of p38a-immunoreactive cells was notably increased, especially in parietal and temporal cortices, where severe neuronal death was known to occur [24] (Fig. 5C). As was in hippocampus, the induction of p38a appeared mainly in activating microglia, which was evidenced by the GSA I-B4 labeling with adjacent section (Fig. 5D and F). At this time, the microglial activation was detected widely in the parietal cortex, where the activated microglia had elongated rod-like shapes (Fig. 5C–F). As was in hippocampus, the immunoreactivity of p38a was concentrated in the nucleus of microglia (Fig. 5E). In contrast to the p38a induction, the p38b induction was not detected in the cortex following the ischemic insult (data not shown).

4. Discussion Here we report the differential induction of p38a and p38b MAPKs in the brain after transient global ischemia. The activation of p38a and p38b MAPKs was closely related to the activation of microglia and astrocytes,

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Fig. 4. Differential expression of p38a and p38b in microglia and astrocytes, respectively. Double immunofluorescent staining was carried out to determine the identity of the p38a- or p38b-positive cells. In hippocampal sections prepared 4 days after global ischemia, the immunoreactivity of p38a (A) was localized in GSA I-B4-positive microglia (B and C), whereas the immunoreactivity of p38b (D) was located in anti-GFAP-positive astrocytes (E and F). The concentrated expression of p38a and 38b was detected in the nucleus of cells (C and F).

respectively, in the post-ischemic brain. Microglia were activated rapidly after ischemia, which occurred within minutes after reperfusion [21]. The induction of p38a was detected in microglia when they began to appear in hippocampus, and continued to occur until 4 days after ischemic insult. On the other hand, p38b induction in astrocytes was delayed and protracted in the post-ischemic brain, which coincided with the time point of astrogliosis that began 2–3 days after ischemic insult [12,23]. The p38 MAPK signaling pathway has been implicated in various pathological conditions of non-neuronal and neuronal cells. The activation of p38 MAPK was critical for the induction of apoptosis in PC12 cells [31], and in glutamate-induced apoptosis in cerebella granule cells [13]. Recent in vivo studies indicated the activation of p38 MAPK pathway in the brain following transient global ischemia [29,27] as well as focal cerebral ischemia [11].

The results of these reports were consistent with our data in that the activation of p38 MAPK in the brain was induced in glia or glia-like cells. Their studies, however, were based on anti-phospho-p38 specific antibody that was unable to differentiate p38a and p38b isoforms. In addition, their examination was limited to the response of p38 MAPK for a short period of time, from 15 min to 1 day [11,27]. While the current work provided the first in vivo evidence supporting the differential function of p38a and p38b in the pathologic brain. Although the detailed mechanism behind such a differential function of p38 MAPK isoforms is not clear at present, it should be obtained by preferential activation of p38 isoforms by the action of specific upstream kinases or via different stress activated pathways [30,6,10]. Transient global ischemia causes severe damage to pyramidal neurons especially in the CA1 region of hip-

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Fig. 5. Immunohistochemical localization of p38a in microglia in cerebral cortex after transient global ischemia. Brain sections including parietal cortices of sham-operated (A), 1 day (B) or 4 days (C-F) after ischemia were stained with anti-p38a antibody (A, B, C and E) or GSA I-B4 (D, F). The p38a immunoreactivity was mildly increased in layers 2–3 at 1 day after ischemia (B) and extended to the whole cortical layers 4 days after ischemia (C). Higher magnification of p38a-positive (E) and GSA I-B4-positive cells (F) in cortex were presented. Note the concentrated expression of p38a in nucleus of microglia (E). Scale bars represent 200 mm for A and 25 mm for E.

pocampus. The pyramidal cell death in the CA1 region takes a delayed time-course, which becomes evident at the light microscopic level 2–4 days after ischemic insult in gerbils and rats [14,24]. This delayed neuronal death in the hippocampus appears to be accompanied by glial cell activation involving both astrocytes and microglia. Activated microglia and astrocytes have been shown to release a variety of cytotoxic agents that lead to neuronal injury [3,7]. Since the activation of p38 MAPK has been implicated in transcription of numerous genes involved in inflammatory processes, including proinflammatory cytokines [1,19] or inducible nitric oxide synthase [4,2], the induction of p38 MAPKs in glia cells in the post-ischemic brain might be responsible for the synthesis of inflammatory mediators. In regard to this, it is intriguing to note that the induction of p38 MAPKs in the postischemic brain was concentrated in the nucleus of microglia and astrocytes (Figs. 2F and 3D). It may also be possible that the induction of p38a and p38b is involved in the expression of genes in the intracellular processes that are important

for cytoskeletal reorganization process occurred during transformation of dormant astrocytes into active ones [19,28].

Acknowledgements This work was supported by a grant of the 21C Frontier Program from KMOST (FG01-0400-010-1-0-0) for J.-K. Lee.

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