Neuroprotection conferred by astrocytes is insufficient to protect animals from succumbing to Japanese encephalitis

Abstract

Astrocytes play a key role in regulating aspects of inflammation and in the homeostatic maintenance of the central nervous system (CNS). However, the role of astrocytes in viral encephalitis mediated inflammation is not well documented. As Japanese encephalitis virus (JEV) infection is localized to neurons and considering the importance of astrocytes in supporting neuronal survival and function, we have exploited an experimental model of Japanese encephalitis (JE) to better understand the role of astrocytes in JE. Suckling mice pups were inoculated with the virus and 2 and 4 days later we analyzed a panel of molecules characteristic of reactive astrogliosis. We show that JEV infection increases the expression of astrocyte-specific glial fibrillary acidic protein (GFAP), the glutamate aspartate transporter (GLAST), glutamate transporter-1 (GLT-1) and ceruloplasmin (CP). The transcript levels of growth factors produced predominantly by activated astrocytes such as nerve growth factor (NGF) and ciliary neurotrophin factor (CNTF) were elevated following JEV infection. The transcript level of brain-derived neurotrophic factor (BDNF) was also elevated following JEV infection. Both NGF and CNTF were capable of preventing ROS mediated neuronal death following in vitro JEV infection to a certain extent. Taken altogether, these data indicate that increased astrogliosis following JEV infection is accompanied by the enhanced ability of astrocytes to detoxify glutamate, inactivate free radical and produce neurotrophic factors that are involved in neuronal protection. However, this elevated physiological state of astrocyte is insufficient in conferring neuroprotection, as infected animals eventually succumb to infection. The response of astrocytes to JE can be amplified to modulate the adaptive response of brain to induce neuroprotection.

Introduction

It has long been known that astrocytes maintain homeostasis in the central nervous system (CNS) to support the survival and information processing functions of neurons. They also respond promptly to CNS infection and help regulate neuroinflammation (Kuhlow et al., 2003). The CNS response to trauma, viral infection, inflammation, excitotoxicity, hypoxia/ischemia, degenerative and dementia-type diseases manifests with accompanied reactive gliosis largely attributed to astrocytes (Bignami and Dahl, 1989, Eng and Ghirnikar, 1994). The astrocyte functional response varies dramatically with the nature of insult (Aschner, 1998a, Norenberg, 1994, Norton et al., 1992, Raivich et al., 1999, Reier and Houle, 1988, Ridet et al., 1997). To maintain homeostasis astrocytes secrete growth factors and trophic factors (Lindholm et al., 1992, Traugott and Lebon, 1988), store energy in the form of glycogen, produce enzymes to detoxify metals and xenobiotics and modulate the immune system (Aschner, 1998a, Aschner, 1998b, Dringen et al., 1993, Dringen et al., 1995, Mucke and Eddleston, 1993, Schousboe et al., 1992, Wilson, 1997). It is suggested that the increased levels of ceruloplasmin (a copper containing dehydrogenase protein is regarded as a marker for activated astrocytes), a protein induced by activated astrocytes, may be a complimentary factor associated with inflammatory conditions (Kuhlow et al., 2003, Lin et al., 2006). All of these properties enable astrocytes to participate in a variety of important physiological processes. A reliable and consistent response of astrocytes to CNS insult involves cytologic and biochemical transformations, including cellular hypertrophy, elevated oxidoreductive enzyme levels, increased glutamine synthetase (GS) and glycogen content, coupled with an increase in immunoreactivity and production of intermediate filaments like glial fibrillary acidic protein (GFAP) and vimentin (Bignami and Dahl, 1989, Eng and Ghirnikar, 1994, Eng et al., 1992, Reier and Houle, 1988). Moreover, glutamate excitotoxicity is increasingly being recognized as an important factor in neuroinflammation and astrocytes are known to play a crucial role in regulating extracellular glutamate. Therefore, the capacity of astrocytes to reduce extracellular levels of glutamate can dramatically impact the extent of neuronal damage following an insult.

Flavivirus are important human pathogens causing variety of diseases ranging from mild febrile illness to severe encephalitis and haemorrhagic fever. Among them, Japanese encephalitis virus (JEV) is an acute zoonotic infection that commonly affects children and is a major cause of acute encephalopathy (Chen et al., 2002). JEV is active over a vast geographic area that includes India, China, Japan and virtually all of South-East Asia. In addition, JEV has been recently isolated from previously non-affected areas of Australia. Approximately 3 billion people live in the JEV endemic area covering much of Asia with nearly 50,000 cases of JE reported each year. Of these, about 10,000 cases results in fatality and a high proportion of survivors have serious neurological and psychiatric sequelae (Kaur and Vrati, 2003).

After entry into the host, JEV generates a rapid inflammatory response, including peripheral leucocytes infiltration (Chaturvedi et al., 1979). Inflammation is accompanied by an increased level of cytokines and chemokines in the serum and cerebrospinal fluid (Mathur et al., 1988). The breakdown of the blood–brain barrier and inflammatory cell infiltration have been observed in JEV infected mice (Hase et al., 1992, Mathur et al., 1992). Astrocytes are part of the blood–brain barrier, a major source of chemokines, and critical effectors of central nervous system inflammation. In vitro studies have shown that besides neuronal cells, astrocytes are also infected by JEV (Chen et al., 2000). Based on neural cell composition and the barrier between the peripheral circulation and CNS, astrocytes might play some role in the transmission of virus from peripheral blood flow into the CNS. As JEV infection in the brain is localized to neurons and as survival of neurons is promoted by astrocytes (Bush et al., 1999, Delaney et al., 1996, Lin et al., 1993, Raff et al., 1993), we chose to investigate the responses of astrocytes to JEV infection. We studied the status of indicators of the astrocytic physiological state. Included were GFAP, GLAST and GLT-1, glutamine synthetase (GS). Ceruloplasmin (CP) and neurotrophic factors nerve growth factor (NGF), brain-derived nerve growth factor (BDNF) and ciliary neurotrophin factor (CNTF) measured at different days post-infection.

Many viruses initiate oxidative stress in infected cells. Oxidative stress occurs in cells when the production of reactive oxygen species (ROS) exceeds intracellular antioxidant defenses (Betteridge, 2000). Higher concentrations of ROS damage cells by oxidizing proteins, DNA, and lipids (Halliwell, 2001, Lombard et al., 2005) or by initiating apoptotic pathways (Ciriolo, 2005). ROS not only play a key role in anti-microbial and anti-tumoral defense, but also contributes to pathogenesis of a wide array of infectious diseases (Halliwell et al., 1992). However, the accumulation of ROS and the role of oxidative stress in the pathogenesis of JEV infection are mostly unclear. In the present communication, we have also studied the role of oxidative stress in neuronal death following JE infection in vitro, as well as the plausible protective role played by neurotrophic factors released from activated astrocytes in modulating the enhanced oxidative stress.