title: Identification of a specific α-synuclein peptide (α-Syn 29-40) capable of eliciting microglial superoxide production to damage dopaminergic neurons.
abstract: Misfolded α-synuclein (α-Syn) aggregates participate in the pathogenesis of synucleinopathies, such as Parkinson's disease. Whereas much is known about how the various domains within full-length α-Syn (FL-α-Syn) contribute to the formation of α-Syn aggregates and therefore to their neurotoxicity, little is known about whether the individual peptides that can be generated from α-syn, possibly as intermediate metabolites during degradation of misfolded α-Syn aggregates, are neurotoxic themselves.
  
  
  
  A series of synthesized α-Syn peptides, corresponding to the locus in FL-α-Syn containing alanine 30, substitution of which with a proline causes a familial form of Parkinson's disease, were examined for their capacity of inducing release of microglial superoxide. The neurotoxicity of these peptides was measured according to their influence on the ability of neuroglial cultures deficient in gp91 (phox) , the catalytic unit of NADPH oxidase (Nox2), or wild-type cultures to take up (3)H-labeled dopamine and on the number of tyrosine hydroxylase-staining-positive neurons. Western blots and confocal images were utilized to analyze membrane translocation of p47 (phox) and p67 (phox) , phosphorylation of p47 (phox) and Erk1/2 kinase, and binding of α-Syn peptides to gp91 (phox) . Activation of brain microglia in mice injected with α-Syn peptides was demonstrated by immunostaining for major histocompatibility complex (MHC)-II along with qPCR for Iba-1 and MHC-II.
  
  
  
  We report α-Syn (29-40) as a specific peptide capable of activating microglial Nox2 to produce superoxide and cause dopaminergic neuronal damage. Administered to mice, this peptide also activated brain microglia to increase expression of MHC-II and Iba-1 and stimulated oxidation reaction. Exploring the underlying mechanisms showed that α-Syn (29-40) peptide triggered Nox2 to generate extracellular superoxide and its metabolite H2O2 by binding to the catalytic unit gp91 (phox) of Nox2; diffusing into cytosol, H2O2 activated Erk1/2 kinase to phosphorylate p47 (phox) and p67 (phox) and further activated Nox2, establishing a positive feedback loop to amplify the Nox2-mediated response.
  
  
  
  Collectively, our study suggests novel information regarding how α-Syn causes neuronal injury, possibly including mechanisms involving abnormal metabolites of α-Syn aggregates.
authors.affiliation: Department of Pathology, University of Washington School of Medicine, Seattle, Washington, 98104, USA.|Neuropharmacology Section, Laboratory of Neurobiology, National Institute of Environmental Health Sciences National Institutes of Health, Research Triangle Park, NC, 27709, USA.
authors.forename: Shijun
authors.initials: S
authors.lastname: Wang
authors.affiliation: Neuropharmacology Section, Laboratory of Neurobiology, National Institute of Environmental Health Sciences National Institutes of Health, Research Triangle Park, NC, 27709, USA.|Institute of Molecular Medicine, National Cheng Kung University, Tainan, 70101, Taiwan.
authors.forename: Chun-Hsien
authors.initials: CH
authors.lastname: Chu
authors.affiliation: Neuropharmacology Section, Laboratory of Neurobiology, National Institute of Environmental Health Sciences National Institutes of Health, Research Triangle Park, NC, 27709, USA.|Department of Laboratory Medicine, Tianjin Haihe Hospital, Tianjin Institute of Respiratory Diseases, Tianjin Medical University, Tianjin, 300350, China.
authors.forename: Mingri
authors.initials: M
authors.lastname: Guo
authors.affiliation: Neuropharmacology Section, Laboratory of Neurobiology, National Institute of Environmental Health Sciences National Institutes of Health, Research Triangle Park, NC, 27709, USA.|Institute of Toxicology, School of Public Health, Shandong University, Jinan, Shandong, 250012, China.
authors.forename: Lulu
authors.initials: L
authors.lastname: Jiang
authors.affiliation: Neuropharmacology Section, Laboratory of Neurobiology, National Institute of Environmental Health Sciences National Institutes of Health, Research Triangle Park, NC, 27709, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Nie
authors.affiliation: Department of Geriatrics, Beijing Tiantan Hospital, Capital Medical University, Beijing, 100050, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Neuropharmacology Section, Laboratory of Neurobiology, National Institute of Environmental Health Sciences National Institutes of Health, Research Triangle Park, NC, 27709, USA.
authors.forename: Belinda
authors.initials: B
authors.lastname: Wilson
authors.affiliation: Department of Pathology, University of Washington School of Medicine, Seattle, Washington, 98104, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Yang
authors.affiliation: Department of Pathology, University of Washington School of Medicine, Seattle, Washington, 98104, USA.
authors.forename: Tessandra
authors.initials: T
authors.lastname: Stewart
authors.affiliation: Neuropharmacology Section, Laboratory of Neurobiology, National Institute of Environmental Health Sciences National Institutes of Health, Research Triangle Park, NC, 27709, USA. hong3@niehs.nih.gov.
authors.forename: Jau-Shyong
authors.initials: JS
authors.lastname: Hong
authors.affiliation: Department of Pathology, University of Washington School of Medicine, Seattle, Washington, 98104, USA. zhangj@uw.edu.|Department of Pathology, Peking University Health Science Center, Beijing, 100083, China. zhangj@uw.edu.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002478:Cells, Cultured; D004195:Disease Models, Animal; D059290:Dopaminergic Neurons / Q000187:drug effects*; D004622:Embryo, Mammalian; D000949:Histocompatibility Antigens Class II / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008840:Microfilament Proteins / Q000378:metabolism; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000378:metabolism; D009457:Neuroglia / Q000187:drug effects* / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D011971:Receptors, Immunologic / Q000172:deficiency / Q000235:genetics; D013481:Superoxides / Q000378:metabolism* / Q000633:toxicity*; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism; D051844:alpha-Synuclein / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*
chemical_list: C491049:Aif1 protein, mouse; D002135:Calcium-Binding Proteins; D000949:Histocompatibility Antigens Class II; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; D010446:Peptide Fragments; C105949:Pirb protein, mouse; D011971:Receptors, Immunologic; D051844:alpha-Synuclein; D013481:Superoxides; D014446:Tyrosine 3-Monooxygenase; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases
keywords: Microglia; Neuronal damage; Superoxide; gp91 phox; α-Synuclein