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Peroxynitrite Generated by Inducible Nitric Oxide Synthase and NADPH Oxidase Mediates Microglial Toxicity to Oligodendrocytes

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Peroxynitrite Generated by Inducible Nitric Oxide Synthase and NADPH Oxidase Mediates Microglial Toxicity to Oligodendrocytes Peroxynitrite generated by inducible nitric oxide synthase and NADPH oxidase mediates microglial toxicity to oligodendrocytes Jianrong Li*, Olivier Baud*, Timothy Vartanian†, Joseph J. Volpe*, and Paul A. Rosenberg*‡ *Department of Neurology, Children’s Hospital, and †Department of Neurology, Beth Israel Deaconess Medical Center, and Program in Neuroscience, Harvard Medical School, Boston, MA 02115 Edited by Richard L. Sidman, Harvard Medical School, Boston, MA, and approved May 29, 2005 (received for review March 28, 2005) Reactive microglia in the CNS have been implicated in the patho- thought to be due to a loss of preOLs and subsequently mature genesis of white matter disorders, such as periventricular leukoma- myelin-producing OLs. The pathogenesis of PVL is believed to lacia and multiple sclerosis. However, the mechanism by which be initiated by two key upstream mechanisms, hypoxia͞ischemia, activated microglia kill oligodendrocytes (OLs) remains elusive. and maternal-fetal infection͞inflammation (10). Recently, it was Here we show that lipopolysaccharide (LPS)-induced death of reported that the cytotoxicity of lipopolysaccharide (LPS) to developing OLs is caused by microglia-derived peroxynitrite, the preOLs was completely dependent on microglia because of the reaction product of nitric oxide (NO) and superoxide anion. Block- activation of the microglial Toll-like receptor 4 signal transduc- ing peroxynitrite formation with nitric oxide synthase inhibitors, tion pathway (11). Astrocytes, OLs, or cortical neurons do not superoxide dismutase mimics, or a decomposition catalyst abro- express this receptor in culture. Thus, the results of this study gated the cytotoxicity. Only microglia, but not OLs, expressed indicate that activation of microglia may contribute to oligoden- inducible NO synthase (iNOS) after LPS challenge; microglia from droglial damage observed in white matter disorders and provide iNOS knockout mice were not cytotoxic upon activation. The a mechanistic link between innate immunity and loss of OLs. The molecular source for superoxide was identified as the superoxide- data are consistent with recent neuropathological findings show- generating enzyme NADPH oxidase. The oxidase was activated ing that the diffuse white matter lesion in PVL is populated with upon LPS exposure, and its inhibition prevented microglial toxicity activated microglia (5). Furthermore, other studies demonstrate toward OLs. Furthermore, microglia isolated from mice deficient in apositiverelationshipbetweenmaternal-fetalinfection,hypoxia͞ the catalytic component of the oxidase, gp91phox, failed to induce ischemia and white matter injury similar to PVL (12–14). cell death. Our results reveal a role for NADPH oxidase in LPS- In this study, we demonstrate that it is peroxynitrite generated induced OL death and suggest that peroxynitrite produced by iNOS from LPS-activated microglia that mediates the death of OLs. and NADPH oxidase in activated microglia may play an important We further determined the molecular sources of peroxynitrite. role in the pathogenesis of white matter disorders. Our data indicate that up-regulation of inducible nitric oxide synthase (iNOS) in microglia but not in preOLs and activation inflammation ͉ lipopolysaccharide ͉ cerebral palsy ͉ reactive nitrogen of the microglial superoxide-generating NADPH oxidase con- species ͉ periventricular leukomalacia tribute to the formation of peroxynitrite that is cytotoxic to developing OLs. icroglia are the resident macrophage-like cells of the CNS. MThey play a pivotal role in the innate immune response of Materials and Methods CNS and are the first line of defense against microorganism Cell Cultures, Cell Treatment, Free Radical Accumulation Measure- invasion and injury (1). Localized activation of microglia, how- ment, and NADPH Diaphorase Histology. Primary OLs and microglia ever, has also been implicated in the pathogenesis of a number were prepared from mixed glial cultures of newborn Sprague– of neurological diseases and disorders, including ischemia; Dawley rat forebrains by using a selective detachment procedure AIDS-associated dementia (2); chronic neurodegenerative dis- with modifications (15). Microglia were isolated by shaking eases (3), such as Alzheimer’s disease, Parkinson’s disease, and flasks containing mixed glia for1hat200rpmonanorbital amyotrophic lateral sclerosis; and white matter disorders, such as shaker (catalog no. 57018-754, VWR Scientific), maintained in multiple sclerosis (4) and periventricular leukomalacia (PVL) DMEM containing 5% FBS and were used the next day. Purified (5). Microglia are extremely responsive to environmental or OLs were cultured in a serum-free basal-defined medium for 7–9 immunological challenges and are the predominant cell type days. The OL cultures were primarily progenitors and precursors ϩ ϩ Ϫ Ϫ producing inflammation-mediated neurodegeneration. Upon (A2B5 ,O4 ,O1 , and myelin basic protein ) and are referred activation, microglia release proinflammatory molecules, such as to as preOLs. Contamination by astrocytes and microglia was cytokines and chemokines, and mediators of cell injury, such as Ͻ2%. Mouse microglia were isolated with the same method as proteases and reactive oxygen species (ROS)͞reactive nitrogen above from mice deficient in Nos2 (iNOS) and cybb (gp91phox). species (6–8). Although necessary for normal functions, activa- See Supporting Materials and Methods, which is published as tion of microglia requires tight regulation to avoid bystander supporting information on the PNAS web site. injury to other CNS cells. The identification of primary toxic factors, localization of their molecular sources, and the elucida- tion of signaling pathways associated with microglial overacti- This paper was submitted directly (Track II) to the PNAS office. vation are therefore of paramount importance to our under- Abbreviations: DPI, diphenyleneiodonium; FeTMPyP, 5,10,15,20-tetrakis(N-methyl-4Ј-pyri- standing of cellular injury in the CNS. dyl)porphyrinato iron III; iNOS, inducible nitric oxide synthase; L-NMMA, N(G)-monometh- PVL is the lesion of cerebral white matter that underlies most yl-L-arginine monoacetate; LPS, lipopolysaccharide; MnTMPyP, manganese(III) tetrakis(N- methyl-2-pyridyl)porphyrin; OL, oligodendrocyte; preOL, premyelinating OL; PVL, cases of cerebral palsy developing in premature infants (9). It is periventricular leukomalacia; ROS, reactive oxygen species; SOD, superoxide dismutase. characterized by focal necrotic lesions in the deep white matter ‡To whom correspondence should be addressed at: Department of Neurology, Children’s and by diffuse injury to premyelinating oligodendrocytes (pre- Hospital, 300 Longwood Avenue, Boston, MA 02115. E-mail: paul.rosenberg@tch. OLs) and possibly axons in the central cerebral white matter. The harvard.edu. diffuse lesion is followed by subsequent hypomyelination, © 2005 by The National Academy of Sciences of the USA 9936–9941 ͉ PNAS ͉ July 12, 2005 ͉ vol. 102 ͉ no. 28 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0502552102 Downloaded by guest on September 25, 2021 Two different types of microglia plus preOL cocultures were used. Microglia were either plated together or placed into transwells above the preOL layer. Cell death was induced by exposure to LPS (Escherichia coli O111:B4, Sigma) in the presence of various reagents as specified in the figure legends. After 24–48 h, cell survival was evaluated. Accumulation of nitrite in the medium was determined by the Greiss reaction. Free radical generation was evaluated with dichlorohydrofluo- rescin diacetate and dihydrorhodamine 123 (15). NADPH di- aphorase was stained as described in Supporting Materials and Methods. Cell Survival Determination. Survival of preOLs was determined by counting O4ϩ cells with normal nuclei. Briefly, cell cultures were treated in triplicate as specified in the figure legends for 24–48 h. After being washed with PBS and fixed with 4% paraformal- Fig. 1. LPS-activated microglia were cytotoxic to preOLs. LPS-induced, mi- dehyde, cells were immunostained for O4. Five random fields croglia-dependent killing of preOLs in preOL plus microglia cocultures was blocked by the NOS inhibitor L-NMMA but not by the ␣-amino-3-hydroxy-5- were counted in each coverslip under ϫ200 magnification, with ͞ Ͼ methyl-4-isoxazolepropionic acid kainate receptor antagonist NBQX. preOL a total of 1,000 cells counted under control conditions. Cell plus microglia cocultures were treated with or without 1 ␮g͞ml LPS in the Ϯ survival is expressed as mean SD. presence of 1 mM L-NMMA, 10 ␮M 1400W, or 100 ␮M NBQX for 48 h. (A) Survival of preOLs was evaluated by immunocytochemistry (red, O4; blue, Translocation of p67phox Assay. Cells were treated with or without bisbenzamide). (Scale bar, 10 ␮m.) (B) Survival of preOLs was evaluated by cell LPS for 18–24 h and collected with a hypotonic buffer (20 mM counting (mean Ϯ SEM). NO production was assayed by the Greiss reaction. Results represent one of three independent experiments with similar results. Hepes, pH 7.5͞10 mM KCl͞1.5 mM MgCl2͞1 mM EGTA͞1mM EDTA͞0.5 mM phenylmethanesulfonyl fluoride͞1 ␮g/ml apro- tinin͞1 ␮g/ml pepstatin͞2 ␮g/ml leupeptin). Cells were disrupted of microglia, LPS did not have any effect on preOL survival. by five cycles of freezing–thawing with liquid nitrogen. After However, in the presence of transwells containing microglia, LPS centrifugation at 10,000 ϫ g for 10 min, plasma membranes were NEUROSCIENCE A obtained by ultracentrifugation of the supernatant
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