Tumor necrosis factor receptor-1-induced neuronal death by TRADD contributes to the pathogenesis of Japanese encephalitis

While a number of studies have documented the neurotropism of Japanese encephalitis virus (JEV), little is known regarding the molecular mechanism of neuronal death following viral infection. The tumor necrosis factor receptor (TNFR)-associated death domain (TRADD) has been suggested to be the crucial signal adaptor that mediates all intracellular responses from TNFR-1. Using mouse (Neuro2a) and human (SK-N-SH) neuroblastoma cell lines, we have shown that the altered expression of TNFR-1 and TRADD following JEV infection regulates the downstream apoptotic cascades. Activation of TRADD led to mitochondria-mediated neuronal apoptosis. As TRADD-knockout animals or deficient cell lines are unavailable, it has been difficult to definitively address the physiological role of TRADD in diseases pathology following JEV infection. We circumvented this problem by silencing TRADD expression with small-interfering RNA (siRNA) and have found that TRADD is required for TNFR-1-initiated neuronal apoptosis following in vitro infection with JEV. Interestingly, siRNA against TRADD also decreased the viral load in Neuro2a cells. Furthermore, siRNA against TRADD increased the survival of JEV-infected mice by altering the expression of pro apoptotic versus antiapoptotic molecules. These studies show that the engagement of TNFR-1 and TRADD following JEV infection plays a crucial role in neuronal apoptosis.     

Minocycline neuroprotects, reduces microglial activation, inhibits caspase 3 induction, and viral replication following Japanese encephalitis

Minocycline is broadly protective in neurological disease models featuring inflammation and cell death and is being evaluated in clinical trials. Japanese encephalitis virus (JEV) is one of the most important causes of viral encephalitis worldwide. There is no specific treatment for Japanese encephalitis (JE) and no effective antiviral drugs have been discovered. Studies indicate that JE involves profound neuronal loss as well as secondary inflammation caused because of cell death. Minocycline is a semisynthetic second-generation tetracycline that exerts anti-inflammatory and antiapoptotic effects that are completely separate from its antimicrobial action. Because tetracycline treatment is clinically well tolerated, we investigated whether minocycline protects against experimental model of JE. Intravenous inoculation of GP78 strain of JEV in adult mice results in lethal encephalitis and caused primarily because of neuronal death and secondary inflammation caused because of cell death. Minocycline confers complete protection in mice following JEV infection ( p  < 0.0001). Neuronal apoptosis, microglial activation, active caspase activity, proinflammatory mediators, and viral titer were markedly decreased in minocycline-treated JEV infected mice on ninth day post-infection. Treatment with minocycline may act directly on brain cells, because neuronal cell line Neuro2a were also salvaged from JEV-induced death. Our data suggest that minocycline may be a candidate to consider in human clinical trials for JE patients.     

Neuropathogenesis of Japanese Encephalitis in a Primate Model

Background

Japanese encephalitis (JE) is a major cause of mortality and morbidity for which there is no treatment. In addition to direct viral cytopathology, the inflammatory response is postulated to contribute to the pathogenesis. Our goal was to determine the contribution of bystander effects and inflammatory mediators to neuronal cell death.

Methodology/Principal Findings

Material from a macaque model was used to characterize the inflammatory response and cytopathic effects of JE virus (JEV). Intranasal JEV infection induced a non-suppurative encephalitis, dominated by perivascular, infiltrates of mostly T cells, alongside endothelial cell activation, vascular damage and blood brain barrier (BBB) leakage; in the adjacent parenchyma there was macrophage infiltration, astrocyte and microglia activation. JEV antigen was mostly in neurons, but there was no correlation between intensity of viral infection and degree of inflammatory response. Apoptotic cell death occurred in both infected and non-infected neurons. Interferon-α, which is a microglial activator, was also expressed by both. Tumour Necrosis Factor-α, inducible nitric oxide synthase and nitrotyrosine were expressed by microglial cells, astrocytes and macrophages. The same cells expressed matrix metalloproteinase (MMP)-2 whilst MMP-9 was expressed by neurons.

Conclusions/Significance

The results are consistent with JEV inducing neuronal apoptotic death and release of cytokines that initiate microglial activation and release of pro-inflammatory and apoptotic mediators with subsequent apoptotic death of both infected and uninfected neurons. Activation of astrocytes, microglial and endothelial cells likely contributes to inflammatory cell recruitment and BBB breakdown. It appears that neuronal apoptotic death and activation of microglial cells and astrocytes play a crucial role in the pathogenesis of JE.     

Death domain complex of the TNFR-1, TRADD,and RIP1 proteins for death-inducing signaling

Apoptosis can be induced by an extrinsic pathway involving the ligand-mediated activation of death receptors such as tumor necrosis factor receptor-1 (TNFR-1). TNFR-1-associated death domain (TRADD) protein is an adapter molecule that bridges the interaction between TNFR-1 and receptor-interacting serine/threonine-protein kinase 1 (RIP1). However, the molecular mechanism of the complex formation of these proteins has not yet been identified. Here, the binding among TNFR-1, TRADD, and RIP1 was identified using a GST pull-down assay and Biacore biosensor experiment. This study showed that structural characterization and formation of the death-signaling complex could be predicted using TNFR-1, TRADD, and RIP1. In addition, we found that the structure-based mutations of TNFR-1 (P367A and P368A), TRADD (F266A), and RIP1 (M637A and R638A) disrupted formation of the death domain (DD) complex and prevented stable interactions among those DDs.     

Caspase 3-dependent cell death of neurons contributes to the pathogenesis of West Nile virus encephalitis

West Nile virus (WNV) is a neurotropic, arthropod-borne flavivirus that has become a significant global cause of viral encephalitis. To examine the mechanisms of WNV-induced neuronal death and the importance of apoptosis in pathogenesis, we evaluated the role of a key apoptotic regulator, caspase 3. WNV infection induced caspase 3 activation and apoptosis in the brains of wild-type mice. Notably, congenic caspase 3(-/-) mice were more resistant to lethal WNV infection, although there were no significant differences in the tissue viral burdens or the kinetics of viral spread. Instead, decreased neuronal death was observed in the cerebral cortices, brain stems, and cerebella of caspase 3(-/-) mice. Analogously, primary central nervous system (CNS)-derived neurons demonstrated caspase 3 activation and apoptosis after WNV infection, and treatment with caspase inhibitors or a genetic deficiency in caspase 3 significantly decreased virus-induced death. These studies establish that caspase 3-dependent apoptosis contributes to the pathogenesis of lethal WNV encephalitis and suggest possible novel therapeutic targets to restrict CNS injury.     

A role for tumor necrosis factor receptor-2 and receptor-interacting protein in programmed necrosis and antiviral responses

Members of the tumor necrosis factor (TNF) receptor (TNFR) superfamily are potent regulators of apoptosis, a process that is important for the maintenance of immune homeostasis. Recent evidence suggests that TNFR-1 and Fas and TRAIL receptors can also trigger an alternative form of cell death that is morphologically distinct from apoptosis. Because distinct molecular components including the serine/threonine protein kinase receptor-interacting protein (RIP) are required, we have referred to this alternative form of cell death as "programmed necrosis." We show that TNFR-2 signaling can potentiate programmed necrosis via TNFR-1. When cells were pre-stimulated through TNFR-2 prior to subsequent activation of TNFR-1, enhanced cell death and recruitment of RIP to the TNFR-1 complex were observed. However, TNF-induced programmed necrosis was normally inhibited by caspase-8 cleavage of RIP. To ascertain the physiological significance of RIP and programmed necrosis, we infected Jurkat cells with vaccinia virus (VV) and found that VV-infected cells underwent programmed necrosis in response to TNF, but deficiency of RIP rescued the infected cells from TNF-induced cytotoxicity. Moreover, TNFR-2-/- mice exhibited reduced inflammation in the liver and defective viral clearance during VV infection. Interestingly, death effector domain-containing proteins such as MC159, E8, K13, and cellular FLIP, but not the apoptosis inhibitors Bcl-xL, p35, and XIAP, potently suppressed programmed necrosis. Thus, TNF-induced programmed necrosis is facilitated by TNFR-2 signaling and caspase inhibition and may play a role in controlling viral infection.     

Reovirus-induced apoptosis requires both death receptor- and mitochondrial-mediated caspase-dependent pathways of cell death

Apoptosis plays an important role in the pathogenesis of many viral infections. Despite this fact, the apoptotic pathways triggered during viral infections are incompletely understood. We now provide the first detailed characterization of the pattern of caspase activation following infection with a cytoplasmically replicating RNA virus. Reovirus infection of HEK293 cells results in the activation of caspase-8 followed by cleavage of the pro-apoptotic protein Bid. This initiates the activation of the mitochondrial apoptotic pathway leading to release of cytochrome c and activation of caspase-9. Combined activation of death receptor and mitochondrial pathways results in downstream activation of effector caspases including caspase-3 and caspase-7 and cleavage of cellular substrates including PARP. Apoptosis is initiated by death receptor pathways but requires mitochondrial amplification producing a biphasic pattern of caspase-8, Bid, and caspase-3 activation.     

Mutational analysis and NMR studies of the death domain of the tumor necrosis factor receptor-1

Tumor necrosis factor receptor-1 (TNFR-1) death domain (DD) is the intracellular functional domain responsible for the receptor signaling activities. To understand the transduction mechanism of TNFR-1 signaling we performed structural and functional analysis of the TNFR-DD. The secondary structure of the TNFR-DD shows that it consists of six anti-parallel alpha-helices. The determination of the topological fold and an extensive mutagenesis analysis revealed that there are two opposite faces that are involved in self-association and interaction with the TRADD death domain. Interestingly, the same critical residues in TNFR-DD are involved in both interactions. There is a good correlation between the binding activities of the mutant proteins and their cytotoxic activities. These results provide important insight into the molecular interactions mediating TNFR-DD self-association and subsequent recruitment of TRADD in the signaling activity of TNFR-1.     

Novel strategy for treatment of viral central nervous system infection by using a cell-permeating inhibitor of c-Jun N-terminal kinase

Viral encephalitis is a major cause of morbidity and mortality worldwide, yet there is no proven efficacious therapy for most viral infections of the central nervous system (CNS). Many of the viruses that cause encephalitis induce apoptosis and activate c-Jun N-terminal kinase (JNK) following infection. We have previously shown that reovirus infection of epithelial cell lines activates JNK-dependent apoptosis. We now show that reovirus infection resulted in activation of JNK and caspase-3 in the CNS. Treatment of reovirus-infected mice with a cell-permeating peptide that competitively inhibits JNK activity resulted in significantly prolonged survival of intracerebrally infected mice following an otherwise lethal challenge with T3D (100 x 50% lethal dose). Protection correlated with reduced CNS injury, reduced neuronal apoptosis, and reduced c-Jun activation without altering the viral titer or viral antigen distribution. Given the efficacy of the inhibitor in protecting mice from viral encephalitis, JNK inhibition represents a promising and novel treatment strategy for viral encephalitis.     

Icam-1 participates in the entry of west nile virus into the central nervous system

Determining how West Nile virus crosses the blood-brain barrier is critical to understanding the pathogenesis of encephalitis. Here, we show that ICAM-1(-/-) mice are more resistant than control animals to lethal West Nile encephalitis. ICAM-1(-/-) mice have a lower viral load, reduced leukocyte infiltration, and diminished neuronal damage in the brain compared to control animals. This is associated with decreased blood-brain barrier leakage after viral infection. These data suggest that ICAM-1 plays an important role in West Nile virus neuroinvasion and that targeting ICAM-1 signaling may help control viral encephalitis.     

Cellular Inflammatory Response to Flaviviruses in the Central Nervous System of a Primate Host

Flaviviruses such as tick-borne encephalitis virus, Japanese encephalitis virus, West Nile virus, and St. Louis encephalitis virus are important neurotropic human pathogens, typically causing a devastating and often fatal neuroinfection. Flaviviruses induce neuroinflammation with typical features of viral encephalitides, including inflammatory cell infiltration, activation of microglia, and neuronal degeneration. Development of safe and effective live-virus vaccines against neurotropic flavivirus infections demands a detailed knowledge of their neuropathogenesis in a primate host that is evolutionarily close to humans. Here, we used computerized morphometric analysis to quantitatively assess the cellular inflammatory responses in the central nervous system (CNS) of rhesus monkeys infected with three antigenically divergent attenuated flaviviruses. The kinetics, spatial pattern, and magnitude of microglial activation, trafficking of T and B cells, and changes in T cell subsets within the CNS define unique phenotypic signatures for each of the three viruses. Our results provide a benchmark for investigation of cellular inflammatory responses induced by attenuated flaviviruses in the CNS of primate hosts and provide insight into the neuropathogenesis of flavivirus encephalitis that might guide the development of safe and effective live-virus vaccines. (J Histochem Cytochem 57:973–989, 2009)     

Activation of microglia by borna disease virus infection: in vitro study

Neonatal Borna disease virus (BDV) infection of the rat brain is associated with microglial activation and damage to the certain neuronal populations. Since persistent BDV infection of neurons in vitro is noncytolytic and noncytopathic, activated microglia have been suggested to be responsible for neuronal cell death in vivo. However, the mechanisms of activation of microglia in neonatally BDV-infected rat brain have not been investigated. To address these issues, activation of primary rat microglial cells was studied following exposure to purified BDV or to persistently BDV-infected primary cortical neurons or after BDV infection of primary mixed neuron-glial cultures. Neither purified virus nor BDV-infected neurons alone activated primary microglia as assessed by the changes in cell shape or production of the proinflammatory cytokines. In contrast, in the BDV-infected primary mixed cultures, we observed proliferation of microglia cells that acquired the round morphology and expressed major histocompatibility complex molecules of classes I and II. These manifestations of microglia activation were observed in the absence of direct BDV infection of microglia or overt neuronal toxicity. In addition, compared to uninfected mixed cultures, activation of microglia in BDV-infected mixed cultures was associated with a significantly greater lipopolysaccharide-induced release of tumor necrosis factor alpha, interleukin 1beta, and interleukin 10. Taken together, the present data are the first in vitro evidence that persistent BDV infection of neurons and astrocytes rather than direct exposure to the virus or dying neurons is critical for activating microglia.     

Japanese encephalitis virus infects neural progenitor cells and decreases their proliferation

Japanese encephalitis virus (JEV), a common cause of encephalitis in humans, especially in children, leads to substantial neuronal injury. The survivors of JEV infection have severe cognitive impairment, motor and behavioral disorders. We hypothesize that depletion of neural progenitor cells (NPCs) by the virus culminates in neurological sequelae in survivors of Japanese encephalitis (JE). We utilized both in vivo model of JEV infection and in vitro neurosphere cultures to study progressive JEV infection. Cellular infection and cell death was determined by flow cytometry. BrdU administration in animals and in neurospheres was used to determine the proliferative ability of NPCs. JEV leads to massive loss of actively proliferating NPC population from the subventricular zone (SVZ). The ability of JEV infected subventricular zone cells to form neurospheres is severely compromised. This can be attributed to JEV infection in NPCs, which however do not result in robust death of the resilient NPC cells. Instead, JEV suppresses the cycling ability of these cells, preventing their proliferation. JEV primarily targets at a critical postnatal age and severely diminishes the NPC pool in SVZ, thus impairing the process of recovery after the insult. This arrested growth and proliferation of NPCs might have an effect on the neurological consequences in JE survivors.     

Japanese encephalitis virus differentially modulates the induction of multiple pro-inflammatory mediators in human astrocytoma and astroglioma cell-lines

Astrocytes become activated in response to many CNS pathologies. The process of astrocyte activation remains rather enigmatic and results in so-called reactive gliosis, a reaction with specific structural and functional characteristics. Astrocytes play a vital role in regulating aspects of inflammation and in the homeostatic maintenance of the CNS. However, the responses of different human astroglial cell-lines in viral encephalitis mediated inflammation are not well documented. We have shown that Japanese encephalitis virus (JEV) infection causes morphological and functional changes in astrocytic cell-lines. We have demonstrated that besides reactive oxygen species (ROS) JEV infection differentially regulated the induction pattern of IL-6, IL-1 beta and IL-8. IP-10, MCP-1, MIG and RANTES secretions in different astroglial cell-lines. The expression of different proteins such as astrocyte-specific glial fibrillary acidic protein (GFAP), the glutamate aspartate transporter/essential amino acid transporter-1 (GLAST/EAAT-1), glutamate transporter-1/essential amino acid transporter-2 (GLT-1/EAAT-2), Ceruloplasmin and Thioredoxin (TRX) expression level also differ in different human astrocyte cell-lines following infection.     

Tumor necrosis factor receptor-associated death domain protein is involved in the neurotrophin receptor-mediated antiapoptotic activity of nerve growth factor in breast cancer cells

The common neurotrophin receptor p75(NTR) has been shown to initiate intracellular signaling that leads either to cell survival or to apoptosis depending on the cell type examined; however, the mechanism by which p75(NTR) initiates its intracellular transduction remains unclear. We show here that the tumor necrosis factor receptor-associated death domain protein (TRADD) interacts with p75(NTR) upon nerve growth factor (NGF) stimulation. TRADD could be immunodetected after p75(NTR) immunoprecipitation from MCF-7 breast cancer cells stimulated by nerve growth factor. In addition, confocal microscopy indicated that NGF stimulation induced the plasma membrane localization of TRADD. Using a dominant negative form of TRADD, we also show that interactions between p75(NTR) and TRADD are dependent on the death domain of TRADD, thus demonstrating its requirement for binding. Furthermore, the p75(NTR)-mediated activation of NF-kappaB was inhibited by transfection with a dominant negative TRADD, resulting in an inhibition of NGF antiapoptotic activity. These results thus demonstrate that TRADD is involved in the p75(NTR)-mediated antiapoptotic activity of NGF in breast cancer cells.     

Chandipura Virus Induces Neuronal Death through Fas-Mediated Extrinsic Apoptotic Pathway

Chandipura virus (CHPV; genus Vesiculovirus, family Rhabdoviridae) is an emerging tropical pathogen with a case fatality rate of 55 to 75% that predominantly affects children in the age group of 2 to 16 years. Although it has been established as a neurotropic virus causing encephalitis, the molecular pathology leading to neuronal death is unknown. The present study elucidates for the first time the mechanism of cell death in neurons after CHPV infection that answers the basic cause of CHPV-mediated neurodegeneration. Through various cell death assays in vitro and in vivo, a relationship between viral replication within neuron and neuronal apoptosis has been established. We report that expression of CHPV phosphoprotein increases up to 6 h postinfection and diminishes thereafter in neuronal cell lines, signifying the replicative phase of CHPV. Various analyses conducted during the investigation established that CHPV-infected neurons are undergoing apoptosis through an extrinsic pathway mediated through the Fas-associated death domain (FADD) following activation of caspase-8 and -3 and prominent cleavage of poly(ADP-ribose) polymerase (PARP). Knocking down the expression of caspase-3, the final executioner of apoptosis, in a neuronal cell line by endoribonuclease-prepared small interfering RNA (siRNA) validated its pivotal role in CHPV-mediated neurodegeneration by showing reduction in apoptosis after CHPV infection.     

Competitive control of independent programs of tumor necrosis factor receptor-induced cell death by TRADD and RIP1

Stimulation of tumor necrosis factor receptor 1 (TNFR1) can initiate several cellular responses, including apoptosis, which relies on caspases, necrotic cell death, which depends on receptor-interacting protein kinase 1 (RIP1), and NF-kappaB activation, which induces survival and inflammatory responses. The TNFR-associated death domain (TRADD) protein has been suggested to be a crucial signal adaptor that mediates all intracellular responses from TNFR1. However, cells with a genetic deficiency of TRADD are unavailable, precluding analysis with mature immune cell types. We circumvented this problem by silencing TRADD expression with small interfering RNA. We found that TRADD is required for TNFR1 to induce NF-kappaB activation and caspase-8-dependent apoptosis but is dispensable for TNFR1-initiated, RIP1-dependent necrosis. Our data also show that TRADD and RIP1 compete for recruitment to the TNFR1 signaling complex and the distinct programs of cell death. Thus, TNFR1-initiated intracellular signals diverge at a very proximal level by the independent association of two death domain-containing proteins, RIP1 and TRADD. These single transducers determine cell fate by triggering NF-kappaB activation, apoptosis, and nonapoptotic death signals through separate and competing signaling pathways.     

Caspase-2 activation is redundant during seizure-induced neuronal death

Seizure-induced neuronal death may be under the control of the caspase family of cell death proteases. We examined the role of caspase-2 in a model of focally evoked limbic seizures with continuous EEG recording. Seizures were elicited by microinjection of kainic acid into the amygdala of the rat and terminated after 40 min by diazepam. Caspase-2 was constitutively present in brain, mostly within neurons, and was detected in both cytoplasm and nucleus. Cleaved caspase-2 (12 kDa) was detected immediately following seizure termination within injured ipsilateral hippocampus, contiguous with increased Val-Asp-Val-Ala-Asp (VDVADase) activity, a putative measure of activated caspase-2. Expression of receptor interacting protein (RIP)-associated Ich-1-homologous protein with death domain (RAIDD) was increased following seizures, whereas expression of RIP and tumor necrosis factor receptor associated protein with death domain (TRADD), other components thought to be linked to the caspase-2 activation and signaling mechanism, were unchanged. Intracerebroventricular administration of z-VDVAD-fluoromethyl ketone blocked seizure-induced caspase-2 activity but did not alter caspase-8 activity and failed to affect DNA fragmentation or neuronal death. These data support activation of caspase-2 following seizures but suggest that parallel caspase pathways may circumvent deficits in caspase-2 function to complete the cell death process.     

Highly permissive infection of microglial cells by Japanese encephalitis virus: a possible role as a viral reservoir

Japanese encephalitis virus (JEV), a mosquito-borne Flavivirus, is a major cause of acute encephalitis, and neurons have been proposed to be the principle JEV target cells in the central nervous system. However, clinically, infection with JEV leads to increased levels of cytokines and chemokines in the serum and cerebrospinal fluid (CSF) the levels of which correlate with the mortality rate of patients. This research aimed to study the role of microglial cells in JEV infection. Mouse microglial cells (BV-2) supported the replication of JEV with extracellular production of virus by 10 h post-infection, and virus titer reached a maximum (2.55 × 10¹⁰ pfu/ml) by day 3 post-infection. While apoptosis was induced in response to virus infection, no alteration in nitric oxide production was observed. Microglial cells remained productively infected with JEV for up to 16 weeks without significant morphological alterations, and the released virions were infectious to mouse neuroblastoma (NA) cells. The high virus production and long persistence of JEV in microglial cells suggests that these cells may serve as viral reservoirs for the infection of neurons in the CNS.