1 Department of Microbiology and Immunology, Research Center for Infectious Disease, Aichi Medical University School of Medicine, Aichi, Japan
2 Laboratory of Virology, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Aichi, Japan
3 Department of Microbiology, Fukui Medical University School of Medicine, 23-3 Matsuoka-cho, Yoshida-gun, Fukui 910-1193, Japan
Correspondence
Yoshinobu Kimura
ykimura{at}fmsrsa.fukui-med.ac.jp
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ABSTRACT |
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INTRODUCTION |
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A mitogen-activated protein kinase (MAPK) superfamily protein functions as a serine/threonine kinase that regulates intracellular signalling cascades and transmits extracellular stimuli from the plasma membrane into the nucleus. Extracellular signal-regulated kinases, the so-called classical MAPKs, are activated by mitogens and survival factors and transduce signals to promote cell proliferation and survival (Matsuzawa & Ichijo, 2001). In contrast, c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 MAPK become activated by a variety of stress signals encompassing UV irradiation, inflammatory cytokines and oxidative stimuli and are implicated in the induction of apoptotic cell death (Mielke & Herdegen, 2000
; Matsuzawa & Ichijo, 2001
). Intriguingly, dsRNA has also been shown to activate these kinases (Iordanov et al., 2000
). Active JNK phosphorylates c-Jun and activating transcription factor 2 (ATF-2). The JNK signalling pathway stabilizes and activates the pro-apoptotic effector p53 and antagonizes the function of anti-apoptotic mediator Bcl-2 by phosphorylation (Mielke & Herdegen, 2000
). Phosphorylated c-Jun stimulates the transcription of several key target genes, including Fas, the death-inducer ligand (Morishima et al., 2001
). Recent studies in various in vitro experimental systems have provided strong evidence that the JNK signal transduction cascade mediates neuronal apoptosis (Luo et al., 1998
; Morishima et al., 2001
; Putcha et al., 2001
; Whitfield et al., 2001
). Activation of this pathway in vivo, especially in the brain, upon exposure to stress stimuli has not yet been addressed enough (Yang et al., 1997
; Xia et al., 2001
). On the other hand, p38 MAPK regulates gene expression by phosphorylating some transcription factors, including cyclic AMP-responsive element-binding protein, myocyte-enhancer factor 2C and ATF-2 (Mielke & Herdegen, 2000
). Significant function of the p38 MAPK pathway has been reported in the apoptotic cascade in cultured neurons (Kummer et al., 1997
; Horstmann et al., 1998
; Ghatan et al., 2000
; Zou et al., 2002
). Activation of p38 MAPK has also been attributed to axotomy-induced apoptosis of retinal ganglion cells in rats, although the role played by p38 activation in apoptosis regulation differs in a cell-type- or stimulation-dependent manner (Kikuchi et al., 2000
).
To clarify the underlying mechanisms whereby virus infection triggers neuronal apoptosis in the brain, we have investigated the activation kinetics of two apoptosis mediators, JNK/SAPK and p38 MAPK, during the course of lethal acute encephalitis caused by neurovirulent influenza A virus infection (Mori et al., 2002b). Here we show that the JNK/SAPK signal transduction cascade becomes activated in virus-infected neurons, while delayed and widespread activation of p38 MAPK occurs in astrocytes and recruits inflammatory responses in the mouse brain.
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METHODS |
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UV inactivation of virus.
Stock virus suspension was exposed to a 15 W UV lamp at a distance of 30 cm for 30 min at 4 °C with continuous and gentle stirring. After irradiation, infectivity was reduced to <10-7 of the original.
Tissue processing.
Under deep anaesthesia by intraperitoneal administration of 7·2 % chloral hydrate in PBS (0·05 ml g-1 body weight), mice were perfused transcardially with 3·7 % formaldehyde in PBS. Brains were soaked in 20 % sucrose in PBS at 4 °C overnight and frozen at -80 °C. Coronal sections of 14 µm thickness of brain tissue were cut at the midbrain level on a cryostat.
Dual immunofluorescent labelling.
Tissue slices were incubated in 5 % donkey serum (Chemicon International) containing 0·3 % Triton-X in PBS for 20 min and reacted in a mixture of two primary antibodies diluted in 2 % donkey serum containing 0·3 % Triton-X in PBS at 4 °C overnight. The primary antibodies adopted in this study included goat polyclonal anti-influenza A virus antibody (working dilution of 1 : 500; Chemicon International), rabbit polyclonal anti-neuronal nitric oxide synthase (nNOS) antibody (working dilution of 1 : 100; Chemicon International), rabbit polyclonal anti-cleaved caspase-3 (Asp175) antibody (working dilution of 1 : 50; Cell Signalling Technology), rabbit polyclonal anti-JNK antibody (working dilution of 1 : 200; Cell Signalling Technology), rabbit polyclonal anti-p38 MAPK antibody (working dilution of 1 : 400; Santa Cruz), rabbit polyclonal anti-phospho-JNK (Thr183/Tyr185) antibody (working dilution of 1 : 200; Cell Signalling Technology), anti-phospho-c-Jun (Ser63) antibody (working dilution of 1 : 200; Cell Signalling Technology), rabbit polyclonal anti-phospho-p38 MAPK (Thr180/Tyr182) antibody (working dilution of 1 : 200; Cell Signalling Technology), mouse monoclonal anti-glial fibrillary acidic protein (GFAP) antibody cocktail (working concentration of 10 µg ml-1; Pharmingen), goat polyclonal anti-TNF- antibody (working dilution of 1 : 200; Santa Cruz) and rabbit polyclonal anti-Fas ligand antibody (working dilution of 1 : 200; Wako Pure Chemical Industries). Then, tissue sections were incubated in secondary antibodies that had been affinity-purified and absorbed for dual immunolabelling (all diluted 1 : 100 in PBS supplemented with 0·3 % Triton-X; Chemicon International), including rodamine-labelled donkey anti-goat immunoglobulin, fluorescein-labelled donkey anti-rabbit immunoglobulin and rodamine-labelled donkey anti-mouse immunoglobulins. Binding was visualized under a confocal laser scanning microscope.
In situ detection of DNA fragmentation.
DNA fragmentation was detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) method using the ApopTag Direct In Situ Apoptosis Detection kit (Intergen). Dual imaging for virus antigens and TUNEL reactions was carried out as described in a previous report (Mori & Kimura, 2000).
Detection of mRNA for influenza A virus membrane protein 1 (M1) and TNF-.
Total mRNA was extracted from brain tissue at the midbrain level using a mRNA Isolation kit (Roche). cDNA synthesis and successive PCR were carried out with the One-Step RNA PCR kit (AMV) (Takara), according to the manufacturer's instructions. Sequences of primer pairs were as follows: influenza A virus M1, 5'-GAGATCGCACAGAGA-3' and 5'-TCGTTGCATCTGCAC-3' (Urabe et al., 1993); mouse TNF-
, 5'-CCTGTAGCCCACGTCGTAGC-3' and 5'-TTGACCTCAGCGCTGAGTTG-3' (Tucker & Sack, 2001
); and mouse
-actin, 5'-ATCTGGCACCACACCTTCTACA-3' and 5'-GTTTCATGGATGCCACAGGATT-3' (Meyding-Lamadé et al., 1998
). PCR conditions were as follows: 50 °C for 30 min and 94 °C for 2 min, followed by 35 cycles of 94 °C for 30 s, 50 °C for 30 s and 72 °C for 1 min. The final products were separated on 1·2 % agarose gel, stained with ethidium bromide and visualized under a UV lamp. Amplified DNA fragments from influenza A virus M1, TNF-
and
-actin transcripts were expected to be 684, 374 and 567 bp, respectively.
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RESULTS |
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Infection of mouse brain with the R404BP strain of influenza A virus
R404BP virus introduced into the right olfactory bulb spread to the midbrain level of the amygdala, ventral tegmental area, pyramidal layer of the hippocampus, substantia nigra zona compacta and mammillary nucleus on day 5 after infection, as reported in a previous report (Mori et al., 2002b). Double immunolabelling of virus antigens and nNOS, which is a marker for neurons, demonstrated that the virus infected neurons selectively in these brain structures (Fig. 1
ac). Neuronal morphology was retained relatively well on day 5. In situ dual labelling showed that 90 % of virus-infected neurons displayed TUNEL-specific signals (n=50) (Fig. 1d
f). Furthermore, 90 % of infected neurons exhibited immunoreactivity for cleaved caspase-3 (n=50) (Fig. 1g
i). Collectively, these findings demonstrated the occurrence of caspase-dependent neuronal apoptosis upon virus infection. On day 7, infected neurons increased in number (Fig. 2
), began to shrink and lost neurite structures, which showed a process of apoptotic neurodegeneration. In the normal brain of age-matched C57BL/6 mice, the levels of both TUNEL-specific and active caspase-3-specific signals were not detectable.
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DISCUSSION |
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Recently, we reported apoptosis of murine olfactory receptor neurons with activation of the JNK/c-Jun/Fas ligand pathway following intranasal infection with the R404BP strain of influenza A virus (Mori et al., 2002a). In the present study, however, we could not detect upregulated expression of Fas ligand molecules in the mouse brain following infection with the same virus. Alternatively, JNK activation in neurons may lead to induction of BIM, a member of the BH3-only pro-apoptotic subfamily of the Bcl-2 protein family, as well as BAX-dependent cytochrome-c release from mitochondria, which induces caspase activation (Putcha et al., 2001
; Whitfield et al., 2001
). These findings underscore the importance of the JNK cascade in induction of neuronal apoptosis in vivo.
Neuronal apoptosis induced by influenza A virus infection in the brain is multifactorial (Mori & Kimura, 2001). Cytotoxic lymphocytes, especially through the perforin/granzyme system-mediated mechanism, play a pivotal role in killing CNS neurons that are infected with the WSN strain of influenza A virus, as demonstrated in mice with targeted disruption of transporter associated with antigen presentation 1 and perforin genes (Mori et al., 1999
; Mori & Kimura, 2000
). Granzyme B released from effector lymphocytes enters the target cells and activates caspase cascades directly (Trapani et al., 2000
). On the other hand, subacute induction of apoptotic neurodegeneration in the brain of influenza virus-infected perforin-/- mice could be attributable to sustained activation of the JNK pathway (Mori et al., 1999
; Mori & Kimura, 2000
). Prolonged JNK activation has been demonstrated in various apoptotic paradigms (Tobiume et al., 2001
). Thus, influenza virus-induced neuronal apoptosis in the brain involves activation of the intrinsic (i.e. apoptosome) and extrinsic (i.e. death receptors and perforin/granzyme) pathways (Putcha et al., 2001
).
Astrocytes with active p38 MAPK produced TNF- molecules (Fig. 6
), synchronous with the development of clinical symptoms in virus-infected mice (hunching and emaciation). Activation of p38 MAPK in astrocytes mediates IL-1
-signalling and results in post-transcriptional production of TNF-
(Lee et al., 2000
). Interestingly, IL-1
-stimulated activation of the p38 MAPK cascade in cultured astrocytes has been reported in connection with prostaglandin E2 production, which gives another example of the relationship between p38 MAPK and neuroinflammation (Molina-Holgado et al., 2000
). Global forebrain ischaemia leads to neuronal death in the hippocampus, activation of p38 taking place in microglia in the vicinity of the affected area (Walton et al., 1998
). Similar to our findings, delayed induction of p38 MAPK has been noted in reactive astrocytes in the regions of the brain undergoing selective neuronal death induced by kainic acid (Che et al., 2001
). In addition, phosphorylation of p38 MAPK has been detected in glial cells within senile plaques in the brain of patients with Alzheimer's disease (Hensley et al., 1999
). These descriptions are consistent with the present observation that activation of p38 MAPK parallels the neuroinflammatory process during virus infection of the brain. It is expected that in combination with antiviral treatment (Mori et al., 2002b
), a therapeutic intervention against the MAPK pathways, using a JNK inhibitor and a p38 inhibitor (Kikuchi et al., 2000
; Lee et al., 2000
; Mielke & Herdegen, 2000
), may suppress apoptotic neuronal death and neuroinflammation during acute virus encephalitis and minimize neurological sequelae.
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ACKNOWLEDGEMENTS |
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Received 26 February 2003;
accepted 27 May 2003.