Inflammatory effects of highly pathogenic H5N1 influenza virus infection in the CNS of mice.

The A/VN/1203/04 strain of the H5N1 influenza virus is capable of infecting the CNS of mice and inducing a number of neurodegenerative pathologies. Here, we examined the effects of H5N1 on several pathological aspects affected in parkinsonism, including loss of the phenotype of dopaminergic neurons located in the substantia nigra pars compacta (SNpc), expression of monoamines and indolamines in brain, alterations in SNpc microglia number and morphology, and expression of cytokines, chemokines, and growth factors. We find that H5N1 induces a transient loss of the dopaminergic phenotype in SNpc and now report that this loss recovers by 90 d after infection. A similar pattern of loss and recovery was seen in monoamine levels of the basal ganglia. The inflammatory response in lung and different regions of the brain known to be targets of the H5N1 virus (brainstem, substantia nigra, striatum, and cortex) were examined at 3, 10, 21, 60, and 90 d after infection. In each of these brain regions, we found a significant increase in the number of activated microglia that lasted at least 90 d. We also quantified expression of IL-1α, IL-1ß, IL-2, IL-6, IL-9, IL-10, IL-12(p70), IL-13, TNF-α, IFN-γ, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, macrophage colony-stimulating factor, eotaxin, interferon-inducible protein 10, cytokine-induced neutrophil chemoattractant, monocyte chemotactic protein-1, macrophage inflammatory protein (MIP) 1α, MIP-1ß, and VEGF, and found that the pattern and levels of expression are dependent on both brain region and time after infection. We conclude that H5N1 infection in mice induces a long-lasting inflammatory response in brain and may play a contributing factor in the development of pathologies in neurodegenerative disorders.

Method for culturing postnatal substantia nigra as an in vitro model of experimental Parkinson's disease.

One of the most prevalent degenerative disorders of the nervous system is Parkinson's disease. The etiology of this disease is for the most part unknown, although it is posited to arise from an interaction of genetic and environmental factors. Although in vivo animal studies have been used to examine the effects of a number of Parkinson-inducing compounds, there is little information on reliable in vitro methodologies that can recapitulate the previously observed in vivo results. Here, we describe a method for generating mixed and chimeric neuron/glial cultures of postnatal substantia nigra (SN), independent of other monoaminergic nuclei in the ventral midbrain. Since many toxins do not affect regions of the midbrain except the SN, use of whole ventral midbrain from embryos can dilute any measurement of cell death. By specifically culturing ventrolateral midbrain containing the substantia nigra, one can more directly target the effects of dopaminergic toxins. In addition, this method can be used to test potential therapies for amelioration of Parkinson's disease.

Glutathione metabolism and Parkinson's disease.

It has been established that oxidative stress, defined as the condition in which the sum of free radicals in a cell exceeds the antioxidant capacity of the cell, contributes to the pathogenesis of Parkinson disease. Glutathione is a ubiquitous thiol tripeptide that acts alone or in concert with enzymes within cells to reduce superoxide radicals, hydroxyl radicals, and peroxynitrites. In this review, we examine the synthesis, metabolism, and functional interactions of glutathione and discuss how these relate to the protection of dopaminergic neurons from oxidative damage and its therapeutic potential in Parkinson disease.

Alterations in glutathione S-transferase pi expression following exposure to MPP+ -induced oxidative stress in the blood of Parkinson's disease patients.

The major motor symptoms of Parkinson's disease do not occur until a majority of the dopaminergic neurons in the midbrain SNpc have already died. For this reason, it is critical to identify biomarkers that will allow for the identification of presymptomatic individuals. In this study, we examine the baseline expression of the anti-oxidant protein glutathione S-transferase pi (GSTpi) in the blood of PD patients and environmentally- and age-matched controls and compare it to GSTpi levels following exposure to 1-methyl-4-phenylpyridinium (MPP(+)), an agent that has been shown to induce oxidative stress. We find that after 4 h of exposure to MPP(+), significant increases in GSTpi levels can be observed in the leukocytes of PD patients. No changes were seen in other blood components. This suggests that GSTpi and potentially other members of this and other anti-oxidant families may be viable biomarkers for PD.

GSTpi expression mediates dopaminergic neuron sensitivity in experimental parkinsonism.

The cause of 95% of Parkinson's disease (PD) cases is unknown. It is hypothesized that PD arises from an interaction of free-radical-generating agents with an underlying genetic susceptibility to these compounds. Here we use the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of parkinsonism to examine the role of a dual function protein, GSTpi, in dopaminergic neuron death. GSTpi is the only GST family member expressed in substantia nigra neurons. GSTpi reduction by pharmacological blockade, RNA inhibition, and gene targeting increases sensitivity to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, suggesting that differential expression of GSTpi contributes to the sensitivity to xenobiotics in the substantia nigra and may influence the pathogenesis of reactive oxygen species-induced neurological disorders including PD.

Glia cell number modulates sensitivity to MPTP in mice.

Free radical damage has been shown to play a significant role in the pathogenesis of a number of neurodegenerative diseases including Parkinson's disease. One model of experimental parkinsonism is the loss of substantia nigra cells following administration of MPTP. Previously, it has been shown that a number of inbred strains of mice have differential responses to this toxin, and this difference is dependent on glial cells. In this study, the number of glial cells in the substantia nigra pars compacta of C57Bl/6J (MPTP-sensitive) and Swiss Webster (MPTP-resistant) strains of mice was examined. The C57Bl/6J mice have an approximately 50% lower number of GFAP+ and S-100beta glial cells than the Swiss Webster mice. C57Bl/6J mice have a 25% increased number of resident nonactivated microglial cells. To determine whether this difference in cell number has functional significance, we used an in vitro SN culture system that allowed us to manipulate the number of glial cells. When C57Bl/6 neurons were grown on a glial mat plated with twice the number of cells, we were able to rescue the MPTP-sensitive neurons from toxin-induced cell death. This suggests that the number of glial cells in the SNpc may be an important factor in the survival of dopaminergic neurons following exposure to xenobiotics.

Characterization of two novel N-methyl-D-aspartate antagonists: EAA-090 (2-[8,9-dioxo-2,6-diazabicyclo [5.2.0]non-1(7)-en2-yl]ethylphosphonic acid) and EAB-318 (R-alpha-amino-5-chloro-1-(phosphonomethyl)-1H-benzimidazole-2-propanoic acid hydrochloride).

Two novel N-methyl-d-aspartate (NMDA) antagonists with unique chemical structures, EAA-090 (2-[8,9-dioxo-2, 6-diazabicyclo[5.2.0]non-1(7)-en2-yl]ethylphosphonic acid) and EAB-318 (R-alpha-amino-5-chloro-1-(phosphonomethyl)-1H-benzimidazole-2-propanoic acid hydrochloride), were compared with CGS-19755 (Selfotel) in ligand binding, electrophysiology, and neuroprotection assays. CGS-19755, EAA-090 and EAB-318 inhibited [(3)H]3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid binding to NMDA receptors with IC(50) values of 55, 28, and 7.9 nM, respectively. All three compounds decreased the duration of spontaneous synaptic currents and inhibited NMDA-activated currents in rat hippocampal neurons. IC(50) values for inhibition of current induced by 10 microM NMDA were 795, 477, and 69 nM for CGS-19755, EAA-090, and EAB-318, respectively. The NMDA antagonists protected chick embryo retina slices and cultured rat hippocampal and cortical neurons from glutamate- and NMDA-induced neurotoxicity. In experiments in which different NMDA receptor splice variants and subtypes were expressed in Xenopus oocytes, all three antagonists preferentially blocked NMDA-elicited currents mediated by N-methyl-d-aspartate receptor (NR)1 splice variants containing the N-terminal insertion. They also favored NR2A-versus NR2B- or NR2C-containing NMDA receptors, with EAA-090 showing the greatest selectivity. EAA-090 was 10 times more potent at blocking NR2A-versus NR2B- or NR2C-containing NMDA receptors. In addition to being the most potent NMDA antagonist, EAB-318 inhibited alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors. The combination of NMDA and AMPA/kainate block enabled EAB-318 to protect neurons against ischemia induced cell death.