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Research report

Immunolocalization of p38 MAP kinase in mouse brain

a a ,

_*

b c

Masumi Maruyama , Tatsuhiko Sudo

, Yoshitoshi Kasuya , Takashi Shiga ,

d a

Bing-Ren Hu , Hiroyuki Osada

Antibiotics Laboratory, RIKEN, 2-1 Hirosawa Wako, Saitama 351-0198 Japan b

School of Medicine, Chiba University, Chiba, Japan c

Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Japan d

Queen’s Medical Center, Hawaii, USA Accepted 3 October 2000

Abstract

p38 has been implicated to play a critical role in regulating apoptosis in PC12 and cerebellar granule cells, and is inactivated in cultured fetal neurons in response to insulin. Though p38 is activated in microglia after ischemia, the physiological functions of p38 in the brain are not well understood. As a first step to elucidate the physiological functions of p38 in the central nervous system, we raised a polyclonal antibody against p38 and performed immunohistochemical examination to demonstrate the localization of p38 in mouse brain. Strong p38 immunoreactivity was apparent in fiber bundles including the olfactory tract, anterior commissure, corpus callosum, cingulum, internal capsule, stria terminalis, fimbria and alveus hippocampi, fornix, stria medullaris, optic chiasm and optic tract. Although similar regions were stained with both anti-p38 and anti-neurofilament antibodies, intense p38 immunoreactivity was often observed in myelin sheath-like structures but not in axons. This is the first demonstration of the localization of p38 in the central nervous system and provides an anatomical basis for understanding physiological roles of p38.  2000 Elsevier Science B.V. All rights reserved.

Theme: Neurotransmitters, modulators, transporters and receptors

Topic: Signal transduction: gene expression

Keywords: p38 MAP kinase; Immunohistochemistry; Oligodendrocyte; Central nervous system

1. Introduction Northern analysis of human tissues [12], whereas p38g is

expressed only in skeletal muscle [16] and d has limited p38 was first identified as either an anti-inflammatory expression in kidney and lung [13].

drug (CSAID) binding protein [15], a lipopolysaccharide In vitro studies have suggested that p38 regulates gene (LPS) activated kinase [8] or a stress responsive kinase expression by phosphorylating transcription factors, CRE-[21]. p38 is a member of mitogen-activated protein kinases BP1 /ATF2 [19], MEF2C [9], CHOP [25] and a transcrip-(MAPK) and is activated by dual phosphorylation of the tional regulator, Max [27]. Meanwhile p38 phosphorylates conserved TGY motif [8,15,21]. p38 has been implicated cellular kinases such as MAPKAPK2 / 3 [17,21], resulting to play roles in converting extracellular stresses to cellular in hyperphosphorylation of HSP27 to modulate actin responses. Thus far, four members of p38 have been dynamics [7]. Although the functional relevance and identified in the human. Among them, p38a and b have significance in physiological conditions are not well under-been shown to be expressed relatively ubiquitously, by stood, p38 may play a role in stress responses.

More recently, p38 has been implicated to play a critical role in regulating apoptosis in PC12 [26] and cerebellar granule cells [14] by its activation. Conversely, p38 is inactivated in cultured fetal neurons in response to insulin *Corresponding author. Tel.: 181-48-467-9542; fax: 1

81-48-462-[10]. Global forebrain ischemia results in apoptosis in 4669.

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M. Maruyama et al. / Brain Research 887 (2000) 350 –358 351 microglias [24]. Moreover analysis performed in ATF2 chased from CLEA Japan Inc. Under deep anesthesia with null mice has indicated that ATF2 plays a critical role in an intraperitoneal injection of sodium pentobarbital, mice the hippocampal CA3 [20]. These results suggest that p38 were perfused through the left cardiac ventricle with saline may play a critical part in maintenance of central nervous followed by ice-cold 4% paraformaldehyde in 0.1 M system (CNS) through the determination of survival and / phosphate buffered saline (PBS), pH 7.4. The brain was or death in response to extracellular stimuli. then removed and immersion fixed in the same fixative for Here we report the localization of p38 in the adult 12 h at 48C. For paraffin sections, the brain was dehydrated mouse brain as a first step to understanding its physiologi- in graded ethanol, embedded in paraffin, and sectioned at 4 cal functions in the CNS. mm thickness. For cryostat sections, the brain was im-mersed in the series of 5, 15 and 30% sucrose solutions in PBS for cryoprotection, frozen in Tissue Tek O.C.T.

2. Material and methods compound (Miles Inc.), and sectioned at 10mm thickness.

For immunoperoxidase labeling, sections were blocked

2.1. Antibodies by incubation with 10% normal horse serum in PBS for 30

min followed by several washes with PBS. They were then A polyclonal anti-p38 antibody was raised by immuniz- incubated with a primary antibody in PBS for 1 h followed ing rabbits with an affinity purified recombinant by several washes with PBS containing 0.05% Triton glutathione-S-transferase-p38a (GST-p38) [22]. The anti- X-100 (PBST). Then sections were incubated in GST-p38 serum was repeatedly applied to GST-c-Jun [11] biotinylated secondary antibody for 1 h followed by column to remove antibodies against GST and bacterial several washes with PBS. They were then incubated in proteins. Then, partially purified serum was multi-step ABC reagent followed by several washes with PBS. Bound affinity purified by binding to GST-p38 column. Mono- peroxidase was visualized by incubating sections with clonal antibodies against neurofilament 68, 160, 200 and 0.05% diaminobenzidine and 0.01% H O2 2 in 50 mM CNPase were purchased from Sigma-Aldrich, Japan K. K. Tris–HCl, pH 7.4. Control sections were incubated with Alexa 488– and Alexa 594–conjugated second antibodies pre-immune serum. All incubations were performed at were purchased from Molecular Probes. Biotinylated sec- room temperature.

ondary antibodies and ABC reagents were purchased from For immunofluorescence double labeling, sections were

Vector Laboratories. blocked by incubating with 10% normal goat serum in

PBS for 30 min followed by several washes with PBS. 2.2. Immunoblot analysis Then they were incubated with a primary antibody in PBS with 2% normal goat serum for 1 or 3 h followed by Dissected brain tissues for immunoblotting were ob- several washes with PBST. Alexa 488– and Alexa 594– tained from adult mouse and sonicated in buffer containing conjugated second antibodies were diluted 2000-fold and 50 mM Hepes–KOH (pH 7.4), 150 mM NaCl, 5 mM incubated for 1 h in PBS containing 2% goat serum. After EDTA, 5 mM EGTA, 20 mM NaF, 1 mM dithiothreitol, 1 extensive washing with PBS, sections were examined by mM phenylmethylsulfonylfluoride, 1 mM sodium ortho- using fluorescent microscopy (Olympus). Control sections vanadate, 1 mM p-nitrophenylphosphate, 20 mM b- were incubated either with pre-immune serum or antibody glycerophosphate and 1% Triton X-100. Then 100mg of preabsorbed with GST-p38.

protein was loaded in each lane after boiling for 3 min in The terminology of brain areas was mainly after Paxinos sample buffer containing sodium dodecyl sulfate (SDS). and Watson [18] and Franklin and Paxinos [6]

The tissue samples were resolved by 12% SDS–poly-acrylamide gel electrophoresis and transferred onto Im-mobilon (Millipore). The filters were blocked for 1 h in 5%

non-fat dried milk in buffer containing 10 mM Tris–HCl 3. Results (pH 7.8) and 144 mM NaCl (TBS) supplemented with

0.05% Tween-20 (TBST). Then filters were incubated for 3.1. Specificity of the anti-p38 antibody 2 h in 1% non-fat dried milk in TBST containing anti-p38

antibody or pre-immune serum. After several washes, the To study the localization of p38 MAP kinase at the blots were incubated with horseradish–peroxidase conju- protein level, we used a rabbit polyclonal antibody against gated anti-rabbit IgG (Santa Cruz) in 1% non-fat dried recombinant GST-p38. Immunoblotting experiments milk containing TBST for 1 h followed by several washes showed that the anti-p38 antibody but not preimmune with TBST, and developed using SuperSignal (Pierce). serum specifically recognized a single band of about 42 kD in mouse brain extracts and extracts of murine embryonic 1/2

2.3. Immunohistochemistry fibroblasts prepared from E10.5 of progeny of p38

intercrosses [23] (Fig. 1). Moreover, the immunoreactivity Eight-week-old C57BL / 6J male mice (n56) were pur- recognized by the anti-p38 antibody disappeared when

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Fig. 1. Specificity of anti-p38 antibody against brain extracts and extracts of murine embryonic fibroblasts (MEF) prepared from E10.5 progeny of

1/2

p38 intercrosses. 100mg of brain homogenate (lanes 1, 2) and 10mg of MEF extracts (lanes 3–8) were applied for immunoblotting experi-ments. Lanes: 1, 3–5 pre-immune serum was used as 1st antibody (negative control): Lanes: 2, 6–8 anti-p38 antibody recognizes a single

2/2

band of 42 kD except for p38 MEF (lane 8). The positions of molecular mass markers are indicated on the left.

coronal sections were stained either with preimmune serum or with antibody pre-absorbed with GST-p38 (Fig. 2). These results demonstrated the specificity of the anti-p38 antibody.

3.2. Localization of p38 in mouse brain

To determine the localization of p38 in adult mouse brain, the anti-p38 antibody was applied to paraffin-em-bedded sections and cryostat sections, which resulted in the similar patterns (Figs. 3 and 4). Strong p38 immuno-reactivity was observed in fiber bundles, but not in cell bodies or nuclei.

In the telencephalon, strong p38 immunoreactivity was observed in the lateral olfactory tract, anterior commissure, corpus callosum, cingulum, internal capsule, external capsule, stria terminalis, fimbria and alveus hippocampi. In the deeper layers of the neocortex, many fibers running in various directions were also immunoreactive for p38 (Fig. 3B). In the diencephalon, strong immunoreactivity was observed in the fornix, mammilothalamic tract, stria

Fig. 2. Specific recognition by anti-p38 antibody in brain sections. medullaris, fasciculus retroflexus, optic chiasm, and optic

Coronal sections were incubated with either anti-p38 antibody (A), tract. In the brainstem, many fiber bundles were strongly

pre-immune serum (B) or preabsorption of anti-p38 antibody with GST-immunoreactive for p38, including cerebral peduncles, _{p38 (C). dlo, dorsolateral olfactory tract; lo, lateral olfactory tract. Scale} longitudinal fasciculus and transverse fibers of the pons. bar5100mm.

In the cerebellum, the white matter (corpus medullare) was strongly immunoreactive for p38 (Fig. 3D).

It is noteworthy that no fibers in CA1 through dentate were immunoreactive for neurofilaments (Fig. 5A, B). gyrus in the hippocampus were immunoreacitve for p38, Dendrites of Purkinje cells in the molecular layer were not whereas many fibers including mossy fibers in CA3 region immunoreactive for p38 in the cerebellum (Fig. 5C, D).

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M. Maruyama et al. / Brain Research 887 (2000) 350 –358 353

Fig. 3. Distribution of p38 immunoreactivity in the forebrain and the cerebellum in sagittal sections. Intense p38 immunoreactivity is observed in ac, anterior commissure; f, fornix; cc, corpus callosum; cg, cingulum; sm, stria medullaris; fr, fasciculus retroflexus; ox, optic chiasm; lfp, longitudinal fasciculus; tfp, transverse fibers of the pons; wm, white matter in the cerebellum. gcc, genu corpus callosum; MHb, medial habenular nucleus; fr, fasciculus retroflexus; ml, molecular layer; gl, granular layer; Pl, Purkinje layer. (A) to (F) were paraffin-embedded sections and (G) was a cryostat section. Scale bars530mm for (A,E,G), 60mm for (B,C,D) and 150mm for (F).

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Fig. 4. Distribution of p38 immunoreactivity in horizontal and coronal sections. Dense p38 immunoreactivity is observed in dlo, dorsolateral olfactory tract; lo, lateral olfactory tract (A); sm, stria medullaris; cc, corpus callosum (B); alv, alveus hippocampi; bsc, brachium superior colliculus; ec, external capsule; fi, fimbria hippocampus; st, stria terminalis (C) in horizontal sections and cg, cingulum; fr, fasciculus retroflexus (D); fr; ic, internal capsule; opt, optic tract; mt, mammillothalamic tract; cp, cerebral peduncles (E) in coronal section. CPu, caudate putamen; Ld, lambdoid septal zone; MHb, medial habenular nucleus; Rt, reticular thalamic nucleus. Scale bar5150mm.

3.3. Cellular localization of p38 immunoreactivity fiber bundles, it is possible that axons and / or myelin sheaths express p38. To identify p38-immunoreactive Since strong p38 immunoreactivity was observed only in structures at the cellular level, sections were first

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double-M. Maruyama et al. / Brain Research 887 (2000) 350 –358 355

Fig. 5. Absence of p38 immunoreactivity in dentate gyrus and parallel fibers. Horizontal (A,B) and sagittal sections (C,D) were labeled with either anti-p38 (A,C) or anti-neurofilament antibodies (B,D). CA3, area CA3; DG, dentate gyrus; ml, molecular layer; gl, granular layer; Pl, Purkinje layer; wm, white matter. Scale bar5150mm.

labeled with anti-p38 and anti-neurofilament 68, 160 and adult mouse brain. Previous studies concerning p38 ex-180 antibodies. At higher resolution, p38 immunoreactivity pression in the brain are very limited. In the hippocampus was often observed in myelin sheath-like structures sur- CA1, phosphorylated p38 is increased in microglia but not rounding axons (Fig. 6A, C, D, F), but not in axons, which neurons after ischemia [24]. Phosphorylated p38 is also were immunoreactive for neurofilaments (Fig. 6B, C, E, increased in cultured cerebellar granule cells under gluta-F). Localization of p38 in myelin sheath structures was mate-induced apoptosis [14]. This is the first report confirmed by double labeling with anti-p38 and anti- regarding the expression of p38 in the central nervous CNPase antibodies (Fig. 7C, F). These results suggest that system under physiological conditions. Our results demon-in some thick fibers, p38 is predomdemon-inantly expressed demon-in the strated that p38 MAP kinase is highly expressed in fiber myelin sheath but not axons. bundles including the olfactory tract, anterior commissure, corpus callosum, cingulum, internal capsule, stria ter-minalis, fimbria and alveus hippocampi, fornix, stria

4. Discussions medullaris, optic chiasm, and optic tract. Although similar

regions were stained with both anti-p38 and anti-neurofila-We raised a specific anti-p38 MAP kinase antibody and ment antibodies, strong immunoreactivity was often ob-analyzed the localization of p38 immunohistochemically in served in myelin sheath-like structures, but not in axons.

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Fig. 6. Cellular localization of p38 immunoreactivity. Sections were double-labeled with anti-p38 and anti-neurofilament 68, 160 and 180 antibodies. At higher resolution, in the caudate putamen, p38 immunoreactivity (green fluorescence) was often observed in myelin sheath-like structures surrounding axons (A, C, D, F), but not in axons, which were immunoreactive for neurofilaments (red fluorescence, B, C, E, F). Arrowheads mark equivalent positions in A to C or D to F respectively. Scale bar52.5mm.

Although p38 has been shown to be expressed in various rule out the possible existence of a relatively low but tissues and cultured cells [10,12–14,16,24,26], the present functionally sufficient amount of p38 in those cells under results clearly show the preferential distribution, or rather physiological conditions. However, the predominant ex-cell type-specific expression of p38 in the CNS, indicating pression of p38 in myelin sheath-like structures may help that additional, yet unreported, physiological functions can to understand its physiological roles in cytokine-induced

be attributed to p38. expression of iNOS gene in oligodendrocytes [2].

The proinflammatory cytokine, tumor necrosis factor-a Further understanding of the physiological functions and (TNF-a) and nitric oxide (NO) have been reported to be regulation of p38 in the CNS will provide insights into the responsible for several CNS disorders, including inflamma- molecular mechanisms of signal transduction pathways. tory, infectious, traumatic and degenerative diseases [3]. In This subject is currently being studied using transgenic inflammatory conditions, inducible nitric oxide synthase mice produced by gene targeting.

(iNOS) which produces NO, is expressed predominantly in activated astrocytes and microglia [4]. By using a specific p38 inhibitor, SB203580, p38 has been demonstrated to

play an important role in endotoxin-induced cellular Acknowledgements response [5]. Key roles of extracellular signal-regulated

kinase (ERK) and p38 in iNOS gene regulation in primary We thank H. Hama, H. Mizuno and M. Yamamoto for cultured glial cells in response to endotoxin have recently helpful discussions, and R. Sato for technical advice. This been reported [1]. Also, p38 is reported to be highly work was supported by President’s Special Grant from phosphorylated after ischemia in microglia but not neurons RIKEN and a grant from the Bioarchitect Research Project in the hippocampus CA1 region [24]. It is not possible to of RIKEN to T. Sudo.

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M. Maruyama et al. / Brain Research 887 (2000) 350 –358 357

Fig. 7. Colocalization of p38 and CNPase immunoreactivities in myelin sheath-like structures. Sections were double-labeled with anti-p38 (green fluorescence) and anti-CNPase (red fluorescence) antibodies. In the caudate putamen, p38 immunoreactivity was often colocalized in myelin sheath-like structures with CNPase. Colocalization can be seen by the yellow-orange color in C and F. Arrowheads mark equivalent positions in A to C or D to F respectively. Scale bar52.5mm.

Regulation of actin filament dynamics by p38 map kinase-mediated

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M. Maruyama et al. / Brain Research 887 (2000) 350 –358 353

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354 M. Maruyama et al. / Brain Research 887 (2000) 350 –358

3.3. Cellular localization of p38 immunoreactivity

fiber bundles, it is possible that axons and / or myelin

sheaths express p38. To identify p38-immunoreactive

Since strong p38 immunoreactivity was observed only in

structures at the cellular level, sections were first

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double-M. Maruyama et al. / Brain Research 887 (2000) 350 –358 355

labeled with anti-p38 and anti-neurofilament 68, 160 and

adult mouse brain. Previous studies concerning p38

ex-180 antibodies. At higher resolution, p38 immunoreactivity

pression in the brain are very limited. In the hippocampus

was often observed in myelin sheath-like structures sur-

CA1, phosphorylated p38 is increased in microglia but not

rounding axons (Fig. 6A, C, D, F), but not in axons, which

neurons after ischemia [24]. Phosphorylated p38 is also

were immunoreactive for neurofilaments (Fig. 6B, C, E,

increased in cultured cerebellar granule cells under

gluta-F). Localization of p38 in myelin sheath structures was

mate-induced apoptosis [14]. This is the first report

confirmed by double labeling with anti-p38 and anti-

regarding the expression of p38 in the central nervous

CNPase antibodies (Fig. 7C, F). These results suggest that

system under physiological conditions. Our results

demon-in some thick fibers, p38 is predomdemon-inantly expressed demon-in the

strated that p38 MAP kinase is highly expressed in fiber

myelin sheath but not axons.

bundles including the olfactory tract, anterior commissure,

corpus callosum, cingulum, internal capsule, stria

ter-minalis, fimbria and alveus hippocampi, fornix, stria

4. Discussions

medullaris, optic chiasm, and optic tract. Although similar

regions were stained with both anti-p38 and

anti-neurofila-We raised a specific anti-p38 MAP kinase antibody and

ment antibodies, strong immunoreactivity was often

ob-analyzed the localization of p38 immunohistochemically in

served in myelin sheath-like structures, but not in axons.

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356 M. Maruyama et al. / Brain Research 887 (2000) 350 –358

Although p38 has been shown to be expressed in various

rule out the possible existence of a relatively low but

tissues and cultured cells [10,12–14,16,24,26], the present

functionally sufficient amount of p38 in those cells under

results clearly show the preferential distribution, or rather

physiological conditions. However, the predominant

ex-cell type-specific expression of p38 in the CNS, indicating

pression of p38 in myelin sheath-like structures may help

that additional, yet unreported, physiological functions can

to understand its physiological roles in cytokine-induced

be attributed to p38.

expression of iNOS gene in oligodendrocytes [2].

The proinflammatory cytokine, tumor necrosis factor-

a

Further understanding of the physiological functions and

(TNF-a) and nitric oxide (NO) have been reported to be

regulation of p38 in the CNS will provide insights into the

responsible for several CNS disorders, including inflamma-

molecular mechanisms of signal transduction pathways.

tory, infectious, traumatic and degenerative diseases [3]. In

This subject is currently being studied using transgenic

inflammatory conditions, inducible nitric oxide synthase

mice produced by gene targeting.

(iNOS) which produces NO, is expressed predominantly in

activated astrocytes and microglia [4]. By using a specific

p38 inhibitor, SB203580, p38 has been demonstrated to

play an important role in endotoxin-induced cellular

Acknowledgements

response [5]. Key roles of extracellular signal-regulated

kinase (ERK) and p38 in iNOS gene regulation in primary

We thank H. Hama, H. Mizuno and M. Yamamoto for

cultured glial cells in response to endotoxin have recently

helpful discussions, and R. Sato for technical advice. This

been reported [1]. Also, p38 is reported to be highly

work was supported by President’s Special Grant from

phosphorylated after ischemia in microglia but not neurons

RIKEN and a grant from the Bioarchitect Research Project

in the hippocampus CA1 region [24]. It is not possible to

of RIKEN to T. Sudo.

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