Chronic encephalomyelitis virus exhibits cellular tropism and evades pDCs by binding to sialylated integrins as the cell surface receptors.

Theiler's murine encephalomyelitis virus (TMEV) causes a chronic demyelinating disease similar to multiple sclerosis in mice. Although sialic acids have been shown to be essential for TMEV attachment to the host, the surface receptor has not been identified. While type I interferons play a pivotal role in the elimination of the chronic infectious Daniel (DA) strain, the role of plasmacytoid dendritic cells (pDCs) is controversial. We herein found that TMEV binds to conventional DCs but not to pDCs. A glycomics analysis showed that the sialylated N-glycan fractions were lower in pDCs than in conventional DCs, indicating that pDCs are not susceptible to TMEV infection due to the low levels of sialic acid. TMEV capsid proteins contain an integrin recognition motif, and dot blot assays showed that the integrin proteins bind to TMEV and that the viral binding was reduced in the desialylated αX ß2 . αX ß2 protein suppressed TMEV replication in vivo, and TMEV co-localized with integrin αM at the cell membrane and TLR 3 in the cytoplasm, suggesting that αM serves as the viral attachment and entry. These results show that the chronic encephalomyelitis virus utilizes sialylated integrins as cell surface receptors, leading to cellular tropism to evade pDC activation.

Generation of a recombinant Saffold Virus expressing UnaG as a marker for the visualization of viral infection.

BACKGROUND: Saffold virus (SAFV), which belongs to the genus Cardiovirus of the family Picornaviridae, is associated with acute respiratory or gastrointestinal illnesses in children; it is also suspected to cause severe diseases, such as acute flaccid paralysis and aseptic meningitis. However, the understanding of the mechanism of its pathogenicity is still limited due to the many unknowns about its lifecycle; for example, the cellular receptor for its infection remains to be determined. A system to monitor SAFV infection in vitro and in vivo is required in order to accelerate research on SAFV. RESULTS: We generated a recombinant SAFV expressing green fluorescent protein (GFP) or UnaG, a novel fluorescent protein derived from Japanese eel. HeLa cells infected by either GFP or UnaG-expressing SAFV showed a bright green fluorescent signal, enabling convenient monitoring of SAFV infection. However, the expression of GFP but not UnaG was quickly lost during virus passaging due to the difference in genetic stability in the SAFV virus genome; the UnaG gene was stably maintained in the virus genome after at least five passages. CONCLUSIONS: SAFV infection of cultured cells can easily be monitored using UnaG-expressing SAFV, which is superior to GFP in terms of genetic stability in the virus genome. This virus could be a useful tool for SAFV research, such as comparing the susceptibility of various cells to SAFV infection and evaluating the effects of antivirals on SAFV infection in high-throughput screening.

Theilovirus 3C Protease Cleaves the C-Terminal Domain of the Innate Immune RNA Sensor, Melanoma Differentiation-Associated Gene 5, and Impairs Double-Stranded RNA-Mediated IFN Response.

Melanoma differentiation-associated gene 5 (MDA5), a member of the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), has pivotal roles in innate immune responses against many positive-stranded RNA viruses, including picornavirus and coronavirus. Upon engagement with dsRNA derived from viral infection, MDA5 initiates coordinated signal transduction leading to type I IFN induction to restrict viral replication. In this study, we describe a targeted cleavage events of MDA5 by the 3C protease from Theilovirus. Upon ectopic expression of theilovirus 3C protease from Saffold virus or Theiler's murine encephalomyelitis virus but not encephalomyocarditis virus, fragments of cleaved MDA5 were observed in a dose-dependent manner. When enzymatically inactive Theilovirus 3C protease was expressed, MDA5 cleavage was completely abrogated. Mass spectrometric analysis identified two cleavage sites at the C terminus of MDA5, cleaving off one of the RNA-binding domains. The same cleavage pattern was observed during Theilovirus infection. The cleavage of MDA5 by Theilovirus protease impaired ATP hydrolysis, RNA binding, and filament assembly on RNA, resulting in dysfunction of MDA5 as an innate immune RNA sensor for IFN induction. Furthermore, the cleavage-resistant MDA5 mutant against the 3C protease showed an enhanced IFN response during Saffold virus infection, indicating that Theilovirus has a strategy to circumvent the antiviral immune response by cleaving MDA5 using 3C protease. In summary, these data suggest MDA5 cleavage by 3C protease as a novel immune evasive strategy of Theilovirus.

Virus-infection in cochlear supporting cells induces audiosensory receptor hair cell death by TRAIL-induced necroptosis.

Although sensorineural hearing loss (SHL) is relatively common, its cause has not been identified in most cases. Previous studies have suggested that viral infection is a major cause of SHL, especially sudden SHL, but the system that protects against pathogens in the inner ear, which is isolated by the blood-labyrinthine barrier, remains poorly understood. We recently showed that, as audiosensory receptor cells, cochlear hair cells (HCs) are protected by surrounding accessory supporting cells (SCs) and greater epithelial ridge (GER or Kölliker's organ) cells (GERCs) against viral infections. Here, we found that virus-infected SCs and GERCs induce HC death via production of the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Notably, the HCs expressed the TRAIL death receptors (DR) DR4 and DR5, and virus-induced HC death was suppressed by TRAIL-neutralizing antibodies. TRAIL-induced HC death was not caused by apoptosis, and was inhibited by necroptosis inhibitors. Moreover, corticosteroids, the only effective drug for SHL, inhibited the virus-induced transformation of SCs and GERCs into macrophage-like cells and HC death, while macrophage depletion also inhibited virus-induced HC death. These results reveal a novel mechanism underlying virus-induced HC death in the cochlear sensory epithelium and suggest a possible target for preventing virus-induced SHL.

Cochlear supporting cells function as macrophage-like cells and protect audiosensory receptor hair cells from pathogens.

To protect the audiosensory organ from tissue damage from the immune system, the inner ear is separated from the circulating immune system by the blood-labyrinth barrier, which was previously considered an immune-privileged site. Recent studies have shown that macrophages are distributed in the cochlea, especially in the spiral ligament, spiral ganglion, and stria vascularis; however, the direct pathogen defence mechanism used by audiosensory receptor hair cells (HCs) has remained obscure. Here, we show that HCs are protected from pathogens by surrounding accessory supporting cells (SCs) and greater epithelial ridge (GER or Kölliker's organ) cells (GERCs). In isolated murine cochlear sensory epithelium, we established Theiler's murine encephalomyelitis virus, which infected the SCs and GERCs, but very few HCs. The virus-infected SCs produced interferon (IFN)-α/ß, and the viruses efficiently infected the HCs in the IFN-α/ß receptor-null sensory epithelium. Interestingly, the virus-infected SCs and GERCs expressed macrophage marker proteins and were eliminated from the cell layer by cell detachment. Moreover, lipopolysaccharide induced phagocytosis of the SCs without cell detachment, and the SCs phagocytosed the bacteria. These results reveal that SCs function as macrophage-like cells, protect adjacent HCs from pathogens, and provide a novel anti-infection inner ear immune system.

Saffold Cardiovirus Infection in a 2-Year-Old Boy with Acute Pancreatitis.

Saffold cardiovirus (SAFV), first identified in a stool sample in 2007, is thought to be associated with respiratory disease and gastroenteritis. On the other hand, animal experiments suggested that the major viral load, following intraperitoneal inoculation of SAFV in mice, may be detected in the pancreas. However, until now, no cases of SAFV in patients with pancreatitis have been reported. This report presents a unique case in a patient who developed relapsing acute pancreatitis (AP) after hand, foot, and mouth disease, and was suspected to have SAFV-1 infection. A 2-year-old boy was admitted to the hospital because of severe abdominal pain. His serum amylase and lipase levels were elevated. Enhanced computed tomography showed pancreatic swelling and dilation of the main pancreatic duct, leading to a diagnosis of severe AP. The viral genome of SAFV-1 was detected by reverse transcription polymerase chain reaction from fecal samples. Furthermore, the serum neutralization titer for SAFV was elevated during AP, but decreased after 1 year. These findings strongly suggest the patient developed SAFV-1 infection concurrent with AP. Therefore, we propose that a cohort study is required to clarify the relationship between SAFV and AP.

Effect of cysteine mutations in the extracellular domain of CM2 on the influenza C virus replication.

CM2 is the second membrane protein of influenza C virus and possesses three conserved cysteines at residue 1, 6 and 20 in its extracellular domain, all of which are involved in the formation of disulfide-linked oligomers of the molecule. In the present study, to examine the effect of CM2 oligomerization on virus replication, we generated a mutant recombinant virus, rC1620A, in which all three cysteines on CM2 were substituted to alanines. The rC1620A virus was more attenuated than the recombinant wild-type (rWT) virus in cultured cells. The CM2 protein synthesized in rC1620A-infected cells could not apparently be detected as a tetramer and was transported to the cell surface less efficiently than was authentic CM2. The amount of CM2 protein incorporated into the rC1620A virions was comparable to that into the rWT virions, although the main CM2 species in the rC1620A virions was in the form of a dimer. Analyses of one-step grown virions and virus-infected cells could not provide evidence for any difference in growth between rC1620A and rWT. On the other hand, the amount of genome present in VLPs possessing the mutant CM2 (C1620A-VLPs) was approximately 31% of that in VLPs possessing wild-type CM2 (WT-VLPs). The incoming genome from VLPs was less efficiently transported to the nucleus in the C1620A-VLP-infected cells than in WT-VLP-infected cells, leading to reduced reporter gene expression in the C1620A-VLP-infected cells. Taken together, these findings demonstrate that CM2 oligomerization affects the packaging and uncoating processes. Thus, we concluded that disulfide-linked CM2 oligomers facilitate virus growth by affecting the replication processes.

Saffold virus type 3 (SAFV-3) persists in HeLa cells.

Saffold virus (SAFV) was identified as a human cardiovirus in 2007. Although several epidemiological studies have been reported, they have failed to provide a clear picture of the relationship between SAFV and human diseases. SAFV genotype 3 has been isolated from the cerebrospinal fluid specimen of patient with aseptic meningitis. This finding is of interest since Theiler's murine encephalomyelitis virus (TMEV), which is the closely related virus, is known to cause a multiple sclerosis-like syndrome in mice. TMEV persistently infects in mouse macrophage cells in vivo and in vitro, and the viral persistence is essential in TMEV-induced demyelinating disease. The precise mechanism(s) of SAFV infection still remain unclear. In order to clarify the SAFV pathogenicity, in the present study, we studied the possibilities of the in vitro persistent infection of SAFV. The two distinct phenotypes of HeLa cells, HeLa-N and HeLa-R, were identified. In these cells, the type of SAFV-3 infection was clearly different. HeLa-N cells were lyticly infected with SAFV-3 and the host suitable for the efficient growth. On the other hand, HeLa-R cells were persistently infected with SAFV-3. In addition, the SAFV persistence in HeLa-R cells is independent of type I IFN response of host cells although the TMEV persistence in mouse macrophage cells depends on the response. Furthermore, it was suggested that SAFV persistence may be influenced by the expression of receptor(s) for SAFV infection on the host cells. The present findings on SAFV persistence will provide the important information to encourage the research of SAFV pathogenicity.

Glycosylation of CM2 is important for efficient replication of influenza C virus.

CM2 is the second membrane protein of influenza C virus and possesses a conserved motif for N-glycosylation. To investigate the role(s) of CM2 glycosylation in the virus replication, we generated rN11A, a recombinant influenza C virus lacking the glycosylation site. The rN11A virus grew less efficiently than the wild-type (WT) virus, although the biochemical characteristics of the mutant CM2 were similar to those of authentic CM2. The amount of the genome (GFP-vRNA) in the CM2-N11A-virus-like particles (VLPs) was 13% of that found in WT-VLPs. The incoming GFP-vRNA was less efficiently transported to the nucleus in CM2-N11A-VLP-infected cells than WT-VLP-infected cells, leading to the reduced reporter gene expression in CM2-N11A-VLP-infected cells. Thus the glycosylation of CM2 is required for efficient replication of influenza C virus, and the obtained findings confirmed and extended the previous observation that CM2 is involved in the genome packaging and uncoating processes.

Saffold virus, a novel human Cardiovirus with unknown pathogenicity.

Although cardioviruses have been thought to mainly infect rodents, a novel human cardiovirus, designated Saffold virus (SAFV), was identified in 2007. SAFV is grouped with Theiler-like rat virus and Theiler's murine encephalomyelitis virus (TMEV) in the species Theilovirus of the genus Cardiovirus of the family Picornaviridae. Eight genotypes of SAFV have now been identified. SAFV has been isolated from nasal and stool specimens from infants presenting with respiratory and gastrointestinal symptoms as well as from children with nonpolio acute flaccid paralysis; however, the relationship of SAFV to this symptomatology remains unclear. Of note, the virus has also been isolated from the cerebrospinal fluid specimens of patients with aseptic meningitis. This finding is of interest since TMEV is known to cause a multiple sclerosis-like syndrome in mice. The involvement of SAFV in various diseases (e.g., respiratory illness, gastrointestinal illness, neurological diseases, and type I diabetes) is presently under investigation. In order to clarify the pathogenicity of SAFV, additional epidemiological studies are required. Furthermore, identification of the SAFV cellular receptor will help establish an animal model for SAFV infection and help clarify the pathogenesis of SAFV-related diseases. In addition, investigation of the tissue-specific expression of the receptor may facilitate development of a novel picornavirus vector, which could be a useful tool in gene therapy for humans. The study of viral factors involved in viral pathogenicity using a reverse genetics technique will also be important.

The preparation of an infectious full-length cDNA clone of Saffold virus.

The pathogenicity of Saffold virus (SAFV) among humans still remains unclear, although it was identified as a novel human cardiovirus in 2007. In order to encourage the molecular pathogenetic studies of SAFV, we generated an infectious cDNA clone of SAFV type 3 (SAFV-3). The present study demonstrated that the synthesis of the full-length infectious RNA by T7 RNA polymerase was terminated by a homologous sequence motif with the human preproparathyroid hormone (PTH) signal in the SAFV-3 genome. To obtain the infectious RNA using T7 promoter, a variant of T7 RNA polymerase, which fails to recognize the PTH signal, was useful. This study will provide a valuable technical insight into the reverse genetics of SAFV.

Palmitoylation of CM2 is dispensable to influenza C virus replication.

CM2 is the second membrane protein of influenza C virus. The significance of the posttranslational modifications of CM2 remains to be clarified in the context of viral replication, although the positions of the modified amino acids on CM2 have been determined. In the present study, using reverse genetics we generated rCM2-C65A, a recombinant influenza C virus lacking CM2 palmitoylation site, in which cysteine at residue 65 of CM2 was mutated to alanine, and examined viral growth and viral protein synthesis in the recombinant-infected cells. The rCM2-C65A virus grew as efficiently as did the parental virus in cultured HMV-II cells as well as in embryonated chicken eggs. The synthesis and biochemical features of HEF, NP, M1 and mutant CM2 in the rCM2-C65A-infected HMV-II cells were similar to those in the parental virus-infected cells. Furthermore, membrane flotation analysis of the infected cells revealed that equal amount of viral proteins was recovered in the plasma membrane fractions of the rCM2-C65A-infected cells to that in the parental virus-infected cells. These findings indicate that defect in palmitoylation of CM2 does not affect transport and maturation of HEF, NP and M1 as well as CM2 in virus-infected cells, and palmitoylation of CM2 is dispensable to influenza C virus replication.

The anti-apoptotic protein L(*) of Theiler's murine encephalomyelitis virus (TMEV) contains a mitochondrial targeting signal.

L(*) protein of TMEV is out-of-frame with the viral polyprotein from an alternative initiation codon AUG, 13 nucleotides downstream from the authentic polyprotein AUG. Anti-apoptotic activity of L(*) was demonstrated by both 'loss of function' and 'gain of function' experiments. However, the precise mechanism(s) of anti-apoptotic activity of L(*) remains to be clarified. In this study, L(*) was demonstrated to be localized to mitochondria. It was also shown by the GFP fusion protein that N-terminal sequence of L(*) may contain a mitochondrial targeting signal (MTS). Surprisingly, L(*)((5-70))-GFP and L(*)((41-70))-GFP were localized to mitochondria although L(*)((1-70))-GFP was distributed in the cytosol, suggesting L(*) has an MTS between amino acid (AA) positions 41 and 70, and that L(*)((1-4)) inhibits its mitochondrial targeting. Furthermore, L(*)((1-70))-GFP was localized to the mitochondria by co-expression of L(*)((65-156)), indicating that L(*)((65-156)) suppresses the inhibition of mitochondrial targeting by L(*)((1-4)). These results suggest that the intra- or inter-molecular interaction of L(*) regulates its mitochondrial localization. It is also suggested that L(*) may inhibit the intrinsic apoptosis through the localization to mitochondria.

Opposite effects of two nonstructural proteins of Theiler's murine encephalomyelitis virus regulates apoptotic cell death in BHK-21 cells.

Theiler's murine encephalomyelitis virus is divided into two subgroups, TO and GDVII, inducing subgroup-specific diseases. In order to investigate the role(s) of nonstructural proteins of TMEV, L and L(∗), leaders of two subgroups, were separately expressed with or without L(∗) in BHK-21 cells. Expression of L increased the number of apoptotic cells. L(∗)/BHK-21 cells constitutively expressing L(∗) showed the decrease in cell death induced by L. These results suggest that L and L(∗) regulate apoptosis during viral infection and contribute to TMEV subgroup-specific biological activities.

Cytokine/chemokine profile in J774 macrophage cells persistently infected with DA strain of Theiler's murine encephalomyelitis virus (TMEV).

Theiler's murine encephalomyelitis virus (TMEV) is a picornavirus and persists in the spinal cords of mice, followed by inflammatory demyelinating disease. Viral persistence is a key determinant for the TMEV-induced demyelination. Macrophages are thought to serve as the site of TMEV persistence during the chronic demyelinating phase. We previously demonstrated that two nonstructural proteins of TMEV, L and L(*), were important for virus growth in J774.1 macrophage cells. However, the key factors of macrophage cells related to virus persistence and demyelination remain poorly understood. The inflammatory response is heavily dependent on cytokine and chemokine production by cell of both the immune system and the central nervous system (CNS). In this study, we established the macrophage cells persistently infected with DA strain, and then analyzed the cytokine expression pattern in those cells. The present results are the first to demonstrate the up-regulation of B-lymphocyte chemoattractant (BLC) and granulocyte colony-stimulating factor (G-CSF) in the macrophage cells persistently infected with DA strain. Furthermore, up-regulation of interleukin (IL)-10 and down-regulation of interferon (IFN)-alpha 4, IFN-beta, and IFN-gamma were shown in those cells. The data suggest that these cytokines/chemokines may contribute to the virus persistence and the acceleration of TMEV-induced demyelination.

Leader (L) of Theiler's murine encephalomyelitis virus (TMEV) is required for virus growth in a murine macrophage-like cell line.

Theiler's murine encephalomyelitis virus is divided into two subgroups on the basis of their different biological activities. GDVII subgroup strains cause acute and fatal encephalomyelitis in mice, while TO or DA subgroup strains cause non-fatal polioencephalomyelitis in weanling mice followed by virus persistence and demyelination in the spinal cords. Nonstructural leader (L) protein is encoded at the most N-terminus of the polyprotein. The L coding region of TO or DA subgroup strains has another out-of-frame open reading frame, which produces another nonstructural protein, L*. L* protein is reported to be essential for virus growth in macrophage cells. In the present report, we studied the role of L protein in virus growth in macrophage-like cell line, J774-1, by using a series of deletion mutant viruses. In J774-1 cells (the absence of L* protein), the mutant virus [deleting the entire L coding region (Delta L), N-terminal zinc-finger domain (Delta Z), acidic domain (Delta A), or C-terminal serine/threonine (S/T)-rich domain (DeltaS/T)] did not grow. The mutant virus disrupting zinc-finger motif (L(cys)) did not grow, either. However, in L*-expressing J774-1 cells (the presence of L* protein), L(cys), Delta Z and DeltaS/T had a rescue of the growth activity, while Delta L or Delta A had no rescue. The data suggest that L protein is required for virus growth in J774-1 cells and also suggest that the site responsible for virus growth in those cells, is the acidic domain of L protein.

Expression of L* protein of Theiler's murine encephalomyelitis virus in the chronic phase of infection.

The DA strain and other members of the TO subgroup of Theiler's murine encephalomyelitis virus synthesize the L* protein from an alternative initiation codon. L* is considered to play a key role in viral persistence and demyelination in susceptible strains of mice, although this hypothesis is still controversial. By using a mutant virus that expresses FLAG epitope-tagged L*, it was demonstrated previously that L* is expressed exclusively in neurons in vivo in the acute phase of infection in the central nervous system (CNS). However, in the mutant virus, the C-H-C-C zinc-binding motif in the leader protein (L) was disrupted by the insertion of the FLAG epitope, resulting in clearance of the virus from the CNS. Therefore, a further two mutant viruses were newly generated, expressing FLAG epitope-tagged L* in which the C-H-C-C zinc-binding motif within L is spared. Both mutant viruses caused persistence and demyelination successfully in spinal cords and enabled us to identify L* immunohistochemically in the demyelinating lesions.

Postischemic alterations of BDNF, NGF, HSP 70 and ubiquitin immunoreactivity in the gerbil hippocampus: pharmacological approach.

1. We investigated the immunohistochemical alterations of BDNF, NGF, HSP 70 and ubiquitin in the hippocampus 1 h to 14 days after transient cerebral ischemia in gerbils. We also examined the effect of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor pitavastatin against the changes of BDNF, NGF, HSP 70 and ubiquitin in the hippocampus after cerebral ischemia in the hippocampus after ischemia. 2. The transient cerebral ischemia was carried out by clamping the carotid arteries with aneurismal clips for 5 min. 3. In the present study, the alteration of HSP 70 and ubiquitin immunoreactivity in the hippocampal CA1 sector was more pronounced than that of BDNF and NGF immunoreactivity after transient cerebral ischemia. In double-labeled immunostainings, BDNF, NGF and ubiquitin immunostaining was observed both in GFAP-positive astrocytes and MRF-1-positive microglia in the hippocampal CA1 sector after ischemia. Furthermore, prophylactic treatment with pitavastatin prevented the damage of neurons with neurotrophic factor and stress proteins in the hippocampal CA1 sector after ischemia. 4. These findings suggest that the expression of stress protein including HSP 70 and ubiquitin may play a key role in the protection against the hippocampal CA1 neuronal damage after transient cerebral ischemia in comparison with the expression of neurotrophic factor such as BDNF and NGF. The present findings also suggest that the glial BDNF, NGF and ubiquitin may play some role for helping surviving neurons after ischemia. Furthermore, our present study indicates that prophylactic treatment with pitavastatin can prevent the damage of neurons with neurotrophic factor and stress proteins in the hippocampal CA1 sector after transient cerebral ischemia. Thus our study provides further valuable information for the pathogenesis after transient cerebral ischemia.

Regulation of mitochondrial morphology and cell survival by Mitogenin I and mitochondrial single-stranded DNA binding protein.

We found that a mouse homolog of human DNA polymerase delta interacting protein 38, referred to as Mitogenin I in this paper, and mitochondrial single-stranded DNA-binding protein (mtSSB), identified as upregulated genes in the heart of mice with juvenile visceral steatosis, play a role in the regulation of mitochondrial morphology. We demonstrated that overexpression of Mitogenin I or mtSSB increased elongated or fragmented mitochondria in mouse C2C12 myoblast cells, respectively. On the other hand, the silencing of Mitogenin I or mtSSB by RNA interference led to an increase in fragmented or elongated mitochondria in the cells, respectively, suggesting that Mitogenin I and mtSSB are involved in the processes of mitochondrial fusion and fission, respectively. In addition, we showed that the silencing of Mitogenin I resulted in an increase in the number of trypan blue-positive cells and the silencing of mtSSB resulted in an enhancement of the sensitivity of the cells to apoptotic stimulation by etoposide. The present results demonstrated that these proteins play a role in cell survival.

Neuroprotective effect of arundic acid, an astrocyte-modulating agent, in mouse brain against MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) neurotoxicity.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes the damage of dopaminergic neurons as seen in Parkinson's disease. Oxidative stress has been as one of several pathogenic hypotheses for Parkinson's disease. Here we investigated whether arundic acid, an astrocyte-modulating agent, can protect against alterations of nitric oxide synthase (NOS) and superoxide dismutase (SOD) expression on MPTP neurotoxicity in mice, utilizing an immunohistochemistry. For this purpose, anti-tyrosine hydroxylase (TH) antibody, anti-dopamine transporter (DAT) antibody, anti-Cu/Zn-SOD antibody, anti-Mn-SOD antibody, anti-nNOS antibody, anti-eNOS antibody and anti-iNOS antibody were used. The present study showed that the arundic acid had a protective effect against MPTP-induced neuronal damage in the striatum and substantia nigra of mice. The protective effect may be, at least in part, caused by the reductions of the levels of reactive nitrogen (RNS) and oxygen species (ROS) against MPTP neurotoxicity. These results suggest that the pharmacological modulation of astrocyte may offer a novel therapeutic strategy for the treatment of Parkinson's disease. Furthermore, our results provide further evidence that a combination of nNOS inhibitors, iNOS inhibitors and free radical scavengers may be effective in the treatment of neurodegenerative diseases. Thus our present results provide valuable information for the pathogenesis of degeneration of the nigrostriatal dopaminergic neuronal pathway.