Title: NADPH Oxidase Mediates Lipopolysaccharide-induced Neurotoxicity and Proinflammatory Gene Expression in Activated Microglia
Abstract: Parkinson's disease is characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra. We have previously reported that lipopolysaccharide (LPS)-induced degeneration of dopaminergic neurons is mediated by the release of proinflammatory factors from activated microglia. Here, we report the pivotal role of NADPH oxidase in inflammation-mediated neurotoxicity, where the LPS-induced loss of nigral dopaminergic neurons in vivo was significantly less pronounced in NADPH oxidase-deficient (PHOX-/-) mice when compared with control (PHOX+/+) mice. Dopaminergic neurons in primary mensencephalic neuron-glia cultures from PHOX+/+ mice were significantly more sensitive to LPS-induced neurotoxicity in vitro when compared with PHOX-/- mice. Further, PHOX+/+ neuron-glia cultures chemically depleted of microglia failed to show dopaminergic neurotoxicity with the addition of LPS. Neuron-enriched cultures from both PHOX+/+ mice and PHOX-/- mice also failed to show any direct LPS-induced dopaminergic neurotoxicity. However, the addition of PHOX+/+ microglia to neuron-enriched cultures from either strain resulted in reinstatement of LPS-induced dopaminergic neurotoxicity, supporting the role of microglia as the primary source of NADPH oxidase-generated insult and neurotoxicity. Immunostaining for F4/80 in mensencephalic neuron-glia cultures revealed that PHOX-/- microglia failed to show activated morphology at 10 h, suggesting an important role of reactive oxygen species (ROS) generated from NADPH oxidase in the early activation of microglia. LPS also failed to elicit extracellular superoxide and produced low levels of intracellular ROS in microglia-enriched cultures from PHOX-/- mice. Gene expression and release of tumor necrosis factor α was much lower in PHOX-/- mice than in control PHOX+/+ mice. Together, these results demonstrate the dual neurotoxic functions of microglial NADPH oxidase: 1) the production of extracellular ROS that is toxic to dopamine neurons and 2) the amplification of proinflammatory gene expression and associated neurotoxicity. Parkinson's disease is characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra. We have previously reported that lipopolysaccharide (LPS)-induced degeneration of dopaminergic neurons is mediated by the release of proinflammatory factors from activated microglia. Here, we report the pivotal role of NADPH oxidase in inflammation-mediated neurotoxicity, where the LPS-induced loss of nigral dopaminergic neurons in vivo was significantly less pronounced in NADPH oxidase-deficient (PHOX-/-) mice when compared with control (PHOX+/+) mice. Dopaminergic neurons in primary mensencephalic neuron-glia cultures from PHOX+/+ mice were significantly more sensitive to LPS-induced neurotoxicity in vitro when compared with PHOX-/- mice. Further, PHOX+/+ neuron-glia cultures chemically depleted of microglia failed to show dopaminergic neurotoxicity with the addition of LPS. Neuron-enriched cultures from both PHOX+/+ mice and PHOX-/- mice also failed to show any direct LPS-induced dopaminergic neurotoxicity. However, the addition of PHOX+/+ microglia to neuron-enriched cultures from either strain resulted in reinstatement of LPS-induced dopaminergic neurotoxicity, supporting the role of microglia as the primary source of NADPH oxidase-generated insult and neurotoxicity. Immunostaining for F4/80 in mensencephalic neuron-glia cultures revealed that PHOX-/- microglia failed to show activated morphology at 10 h, suggesting an important role of reactive oxygen species (ROS) generated from NADPH oxidase in the early activation of microglia. LPS also failed to elicit extracellular superoxide and produced low levels of intracellular ROS in microglia-enriched cultures from PHOX-/- mice. Gene expression and release of tumor necrosis factor α was much lower in PHOX-/- mice than in control PHOX+/+ mice. Together, these results demonstrate the dual neurotoxic functions of microglial NADPH oxidase: 1) the production of extracellular ROS that is toxic to dopamine neurons and 2) the amplification of proinflammatory gene expression and associated neurotoxicity. The pathogenesis of several neurological disorders, including Parkinson's disease, Alzheimer's disease, multiple sclerosis, and the AIDS dementia complex, has been closely associated with localized inflammatory responses in the brain (1.Dickson D.W. Lee S.C. Mattiace L.A. Yen S.H. Brosnan C. Glia. 1993; 7: 75-83Crossref PubMed Scopus (794) Google Scholar, 2.McGeer P.L. Itagaki S. Boyes B.E. McGeer E.G. Neurology. 1988; 38: 1285-1291Crossref PubMed Google Scholar, 3.Liu B. Hong J.S. J. Pharmacol. Exp. Ther. 2003; 304: 1-7Crossref PubMed Scopus (945) Google Scholar, 4.Sherman M.P. Griscavage J.M. Ignarro L.J. Med. Hypotheses. 1992; 39: 143-146Crossref PubMed Scopus (81) Google Scholar). Glial cell activation and oxidative stress have been frequently observed during the course of these disease processes (5.Cicchetti F. Brownell A.L. Williams K. Chen Y.I. Livni E. Isacson O. Eur. J. Neurosci. 2002; 15: 991-998Crossref PubMed Scopus (336) Google Scholar, 6.Liu B. Gao H.M. Wang J.Y. 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The identification of ROS as a primary factor in LPS-mediated neurodegeneration, the localization of the source of ROS causing the neurotoxic effects, and the elucidation of the signaling pathway driving microglial overactivation is of paramount importance to understanding the molecular mechanisms of the neurodegenerative disease state. In effort to answer these questions, the following group of experiments sought to discern the role of NADPH oxidase in microglia-mediated neurotoxicity as both a source of neurotoxic ROS and as a critical cellular mediator responsible for the amplification of the microglia proinflammatory response. In this paper, we have performed both an in vivo study by nigral injection of LPS and an in vitro study using mesencephalic neuron-glia or enriched microglial cultures from both PHOX-deficient mice and PHOX+/+ control mice in effort to delineate the role of NADPH oxidase-generated ROS in LPS-induced dopaminergic neurotoxicity and to discern the underlying mechanism. Animals—Eight-week-old male (25-30 g) and female (25-30 g) B6.129S6-Cybbtm1Din (PHOX-/-) and C57BL/6J 000664 (PHOX+/+) mice were purchased from Jackson Laboratories (Bar Harbor, ME). B6.129S6-Cybbtm1Din PHOX-/- mice are lacking a functional gp91 protein, the catalytic subunit of the NADPH oxidase complex. The PHOX-/- mutation is maintained in the C57BL/6J 000664 background; therefore, C57BL/6J 000664 (PHOX+/+) mice were used as control animals. Breeding of the mice was performed to achieve accurate timed pregnancy within ± 0.5 days. Reagents—Lipopolysaccharide (strain O111:B4) was purchased from Calbiochem (San Diego, CA). Cell culture ingredients were obtained from Invitrogen. [3H]Dopamine (DA; 28 Ci/mmol) was purchased from PerkinElmer Life Sciences. Monoclonal antibody against neuron-specific nuclear protein (Neu-N) was obtained from Pharmingen (San Diego, CA). The polyclonal antibody against glial fibrillary acidic protein (GFAP) and antibody diluent came from DAKO (Carpinteria, CA). The polyclonal antibody against tyrosine hydroxylase (TH) was a kind gift from Dr. John Reinhard of Glaxo Wellcome (Research Triangle Park, NC). Rat monoclonal antibody raised against F4/80 antigen was purchased from Serotec (Washington, D. C.). Biotinylated horse anti-mouse and goat anti-rabbit secondary antibodies were purchased from Vector Laboratories (Burlingame, CA). 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) was obtained from Calbiochem (La Jolla, CA). TNFα enzyme-linked immunosorbent assay kits were purchased from R & D Systems Inc. (Minneapolis, MN). All other reagents came from Sigma. LPS Injection in Vivo—Male PHOX-/- and PHOX+/+ mice were anesthetized with sodium pentobarbital (80 mg/kg) and positioned in a small animal stereotaxic apparatus. Injection of LPS into substantia nigra (SN) was made using the flowing stereotaxic coordinates, measured from bregma (33.Paxinos G. Franklin K.B.J. The Mouse Brain in Stereotaxic Coordinates. Academic Press, San Diego, CA1997Google Scholar): 3.0 mm posterior, 1.3 mm lateral, and 4.7 mm ventral. LPS (5 μg in 2 μl of saline) was injected into the right side of the SN over a period of 2 min, and the injection needle was kept in place for 2 min after the injection. A control injection of saline alone was made into the left side of SN under the conditions described above. Eight animals were used for each strain. Mesencephalic Neuron-Glia Cultures—Mouse ventral mesencephalic neuron-glia were prepared following a previously described protocol (34.Liu B. Du L. Kong L.Y. Hudson P.M. Wilson B.C. Chang R.C. Abel H.H. Hong J.S. Neuroscience. 2000; 97: 749-756Crossref PubMed Scopus (78) Google Scholar) with modifications. Briefly, ventral mesencephalic tissues dissected from embryonic day 13/14. PHOX+/+or PHOX-/- tissues were dissociated by mild mechanical trituration in minimum essential medium. The cells were plated (5 × 105/well) in 24-well culture plates precoated with poly-d-lysine (20 μg/ml) and cultured using the previously reported procedure (34.Liu B. Du L. Kong L.Y. Hudson P.M. Wilson B.C. Chang R.C. Abel H.H. Hong J.S. Neuroscience. 2000; 97: 749-756Crossref PubMed Scopus (78) Google Scholar). The composition of the mesencephalic neuron-glia cultures was determined by immunocytochemistry. The mouse mesencephalic neuron-glia cultures contained 10-11% F4/80-IR microglia, 40% Neu-N-IR neurons, and 1% TH-IR dopaminergic neurons. The remaining cells were presumed to be astroglia. Mesencephalic Microglia-depleted Cultures—Mesencephalic neuron-glia were seeded at 5 × 105/well in 24-well plates. Microglia were depleted by 0.5 mm l-leucine methyl ester for 72 h. The cultures stained with F4/80 antibody showed less than 0.1% microglia. Mesencephalic Neuron-enriched Cultures—Mesencephalic neuron-glia seeded at 5 × 105/well in 24-well plates were treated with 5 μm cytosine β-d-arabinofuranoside at 48 h. Two days later, the cytosine β-d-arabinofuranoside was removed. The cultures stained with F4/80 and GFAP antibodies showed a purity of >98%. Microglia-enriched Cultures—Primary microglia were prepared from whole brains of 2-day-old control (PHOX+/+) or PHOX-/- pups following a previously described protocol (34.Liu B. Du L. Kong L.Y. Hudson P.M. Wilson B.C. Chang R.C. Abel H.H. Hong J.S. Neuroscience. 2000; 97: 749-756Crossref PubMed Scopus (78) Google Scholar). The cells were seeded at 5× 104/well in 96-well plates for TNFα or superoxide assays. The purity of microglia was >98%. Analysis of Neurotoxicity—The degeneration of dopaminergic neurons was assessed by measuring the ability of cultures to take up [3H]DA and counting the number of TH-IR neurons following immunostaining in mesencephalic neuron-glia cultures. For in vivo study, 24 consecutive brain slices (35-μm thickness), which encompassed the entire substantia nigra, compact, were collected. A normal distribution of the number of TH-immunoreactive (TH-IR) neurons in the substantia nigra, compact was constructed based on the counts of 24 slices from PHOX-/- mutant mice and PHOX+/+ mice. The distribution curves from these two noninjected groups superimpose and show no difference in number and shape of the curves. Eight evenly spaced brain slices from saline or LPS-injected animals were immunostained with an antibody against TH and counted. The distribution of the cell numbers from each animal was matched with the normal distribution curve to correct for errors resulting from the cutting. With stereology equipment, three individuals and the Computer-Assisted Stereology Toolbox (Olympus Danmark A/S, Albertslund, Denmark) system performed counting in a double-blind manner. Conclusions were drawn only when the difference was within 5%. Uptake Assay—The cells were incubated for 20 min at 37 °C with 1 μm [3H]DA in Krebs-Ringer buffer (16 mm NaH2PO4, 16 mm Na2HPO4, 119 mm NaCl, 4.7 mm KCl, 1.2 mm MgSO4, 1.3 mm EDTA, pH 7.4). Nonspecific uptake was measured in the presence of 10 μm mazindol. After the cells were washed (three times) with ice-cold Krebs-Ringer buffer (1 ml/well) and lysed with 1 n NaOH (0.5 ml/well), the lysate was mixed with 15 ml of scintillation fluid, and radioactivity was determined with a liquid scintillation counter. The specific [3H]DA was calculated by subtracting the amount of radioactivity obtained in the presence of mazindol from that obtained in the absence of mazindol. Immunostaining—Neurons were stained with the antibody against Neu-N, for neuronal cell bodies but not for neurites. Microglia were stained with rat monoclonal antibody raised against F4/80 antigen, and astrocytes were stained with the antibody against GFAP, an intermediate filament protein whose synthesis is restricted to astrocytes. Dopamine neurons were detected with the polyclonal antibody against TH. Briefly, the cells were fixed for 20 min at room temperature in 3.7% paraformaldehyde in phosphate-buffered saline (PBS). After washing (two times) with PBS, the cultures were treated with 1% hydrogen peroxide for 10 min. The cultures were again washed (three times) with PBS and then incubated for 40 min with blocking solution (PBS containing 1% bovine serum albumin, 0.4% Triton X-100, and 4% appropriate serum: normal horse serum for Neu-N, or F4/80 and normal goat serum for TH or GFAP staining). The cultures were incubated overnight at 4 °C with the primary antibody diluted in DAKO antibody diluent, and then the cells were washed (three times) for 10 min each time in PBS. The cultures were next incubated for 1 h with PBS containing 0.3% Triton X-100 and the appropriate biotinylated secondary antibody (Neu-N, horse anti-mouse antibody, 1:227; TH or GFAP, goat anti-rabbit antibody, 1:227; F4/80, mouse anti-rat antibody, 1:500). After washing (three times) with PBS, the cultures were incubated for 1 h with the Vectastain ABC reagents diluted in PBS containing 0.3% Triton X-100. After washing (two times) with PBS, the bound complex was visualized by incubating cultures with 3,3′-diaminobenzidine and urea-hydrogen peroxide tablets from Sigma dissolved in water. Color development was terminated by removing the reagents and washing the cultures (two times) with PBS. For cell counting, nine representative areas/well in the 24-well plate were counted under the microscope at 100× magnification. Superoxide Assay—The amount of extracellular superoxide (O2˙-) produced was determined by measuring the superoxide dismutase-inhibitable reduction of tetrazolium salt, WST-1 (35.Peskin A.V. Winterbourn C.C. Clin. Chim. Acta. 2000; 293: 157-166Crossref PubMed Scopus (396) Google Scholar, 36.Liu B. Hong J.S. Methods Mol. Med. 2003; 79: 387-395PubMed Google Scholar, 37.Tan A.S. Berridge M.V. J. Immunol. Methods. 2000; 238: 59-68Crossref PubMed Scopus (295) Google Scholar). Primary microglia were plated at 5 × 104/well in 200 μl of culture medium in 96-well plates and incubated for 24 h at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. The cells were washed twice with Hanks' balanced salt solution (HBSS). To each well, 100 μl of HBSS with or without superoxide dismutase (600 units/ml), 50 μl of vehicle or LPS, and 50 μl of WST-1 (1 mm) in HBSS were added. The cultures were incubated for 30 min at 37 °C and 5% CO2 and 95% air. The absorbance at 450 nm was read with a Spectra Max Plus microtiter plate spectrophotometer (Molecular Devices, Sunnyvale, CA). The amount of superoxide dismutase-inhibitable superoxide was calculated and expressed as a percentage of vehicle-treated control cultures. Intracellular oxidative stress was measured by DCFH Oxidation. DCFH-DA enters cells passively and is deacetylated by esterase to nonfluorescent DCFH. DCFH reacts with ROS to form DCF, the fluorescent product. DCFH-DA was dissolved in methanol at 10 mm and was diluted 500-fold in HBSS to give DCFH-DA at 20 μm. The cells were exposed to DCFH-DA for 1 h and then treated with HBSS containing the corresponding concentrations of LPS for 2 h. The fluorescence was read immediately at wavelengths of 485 nm for excitation and 530 nm for emission on a fluorescence plate reader. The value subtracted by control group was viewed as the increase of intracellular ROS. Real Time RT-PCR Analysis—The level of TNFα gene expression was quantified using real time RT-PCR analysis as described by Walker (38.Walker N.J. J. Biochem. Mol. Toxicol. 2001; 15: 121-127Crossref PubMed Scopus (92) Google Scholar). Briefly, total RNA was isolated from mesencephalic neuron-glia cultures from PHOX-/- and PHOX+/+ mice using Trizol reagent, followed by purification with RNeasy column (Qiagen). Total RNA was reverse transcribed with murine leukemia virus reverse transcriptase and random hexamer primers. The following forward and reverse primers for TNFα were designed using Primer Express software (version 2.0; Applied Biosystems, Foster City, CA): TNFα forward, 5′-GAC CCT CAC ACT CAG ATC ATC TTC T-3′; TNFα reverse, 5′-CCT CCA CTT GGT GGT TTG CT-3′. The SYBR green DNA PCR kit (Applied Biosystems, Foster City, CA) was used for real time PCR analysis. The relative differences in expression between groups were expressed using cycle time values normalized with β-actin, and the relative differences between control and treatment groups were calculated and expressed as relative increases setting control as 100%. TNFα Assay—Release of TNFα was measured with a commercial enzyme-linked immunosorbent assay kit from R & D Systems (Minneapolis, MN), as described previously (6.Liu B. Gao H.M. Wang J.Y. Jeohn G.H. Cooper C.L. Hong J.S. Ann. N. Y. Acad. Sci. 2002; 962: 318-331Crossref PubMed Scopus (381) Google Scholar). Statistical Analysis—The data are expressed as the means ± S.E., and statistical significance was assessed with an analysis of variance followed by Bonferroni's t test using the StatView program (Abacus Concepts, Inc., Berkeley, CA). A value of p < 0.05 was considered statistically significant. PHOX -/- Mice Show Reduced LPS-induced DA Neurotoxicity in the Substantia Nigra in Vivo—To investigate the role of NADPH oxidase-generated ROS in LPS-induced neurotoxicity, LPS (5 μg in 2 μl of saline) was stereotaxically injected into the SN of PHOX+/+ and PHOX-/- mice. Fourteen days later, the brains were removed, and coronal sections through the nigral complex were taken. The sections were immunostained with an antibody against TH to detect dopaminergic neurons. Immunocytochemical analysis indicated that LPS injections caused a greater loss of TH-IR neurons in the SN of PHOX+/+ mice than that of PHOX-/- mice, compared with the corresponding saline-injected sides (Fig. 1A). Cell count analysis revealed a 44% loss of DA neurons in PHOX+/+ controls and 18% loss in PHOX-/- mutants, compared with the corresponding saline-injected control sides (Fig. 1B). These results showed that TH-IR neurons in the substantia nigra of PHOX-/- mutants were less affected by LPS-induced inflammatory insult, compared with PHOX+/+ controls. These findings suggest that NADPH oxidase-generated ROS play an important role in LPS-induced degeneration of dopaminergic neurons in the SN. Neuron-Glia Cultures from PHOX-/- Mice Show Reduced LPS-induced DA Neurotoxicity than Those from PHOX+/+ Mice—To further understand the mechanisms underlying the difference in LPS-induced dopaminergic neurotoxicity between PHOX+/+ and PHOX-/- mice observed in vivo, mixed mesencephalic neuron-glia cultures from PHOX+/+ and PHOX-/- mice were treated with either vehicle or LPS (1-100 ng/ml). Neurotoxicity was assessed by DA uptake, morphological analysis and TH-IR neuron number. The cultures from PHOX+/+ and PHOX-/- mice showed a concentration-dependent decrease in both [3H]DA uptake and the number of tyrosine hydroxylase-immunoreactive (TH-IR) neurons. However, in neuron-glia cultures from PHOX-/- mice, the decrease in DA uptake and the number of TH-IR neurons was significantly less than that in neuron-glia cultures from PHOX+/+ mice (Fig. 2, A and B). Morphological analysis showed that TH-IR neurons in the LPS-treated cultures from PHOX+/+ mice displayed shorter and less elaborate TH-IR dendrites compared with those from PHOX-/- mice (Fig. 2C). This difference in the LPS-induced neurotoxicity of dopaminergic neurons between the cultures from PHOX+/+ and PHOX-/- mice suggests an important role for ROS in LPS-induced neuron death. NADPH oxidase mediates LPS-induced morphological changes associated with the activation of microglia. Earlier work from our laboratory demonstrates that LPS is not directly toxic to dopaminergic neurons and that microglia, rather than astroglia, are pivotal to LPS-induced neurotoxicity (39.Kim W.G. Mohney R.P. Wilson B. Jeohn G.H. Liu B. Hong J.S. J. Neurosci. 2000; 20: 6309-6316Crossref PubMed Google Scholar). To address whether microglia were the source of NADPH oxidase and consequent neurotoxicity for LPS-treated cells, the PHOX+/+ and PHOX-/- mesencephalic neuron-glia, neuron-astrocyte, and neuron-enriched cultures were treated with vehicle or 100 ng/ml LPS for 7 days, respectively. In neuron-glia cultures, LPS caused a significant decrease in DA uptake (Fig. 3A). In neuron-astrocyte cultures, the PHOX+/+ and PHOX-/- mesencephalic neuron-glia were treated with l-leucine methyl ester to chemically deplete the microglia. l-Leucine methyl ester-treated PHOX+/+ and PHOX-/- cultures devoid of microglia did not show significant neurotoxicity (Fig. 3B). The result was further confirmed by the PHOX+/+ and PHOX-/- neuron-enriched cultures treated with LPS (Fig. 3C), wh