title: Prothrombin kringle-2 induces death of mesencephalic dopaminergic neurons in vivo and in vitro via microglial activation.
abstract: We have shown that prothrombin kringle-2 (pKr-2), a domain of human prothrombin distinct from thrombin could activate cultured rat brain microglia in vitro. However, little is known whether pKr-2-induced microglial activation could cause neurotoxicity on dopaminergic (DA) neurons in vivo. To address this question, pKr-2 was injected into the rat substantia nigra (SN). Tyrosine hydroxylase (TH) immunohistochemistry experiments demonstrate significant loss of DA neurons seven days after injection of pKr-2. In parallel, pKr-2-activated microglia were detected in the SN with OX-42 and OX-6 immunohistochemistry. Reverse transcription PCR and double-label immunohistochemistry revealed that activated microglia in vivo exhibit early and transient expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and several proinflammatory cytokines. The pKr-2-induced loss of SN DA neurons was partially inhibited by the NOS inhibitor N(G)-nitro-L-arginine methyl ester hydrochloride, and the COX-2 inhibitor DuP-697. Extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase were activated in the SN as early as 1 hr after pKr-2 injection, and localized within microglia. Inhibition of these kinases led to attenuation of mRNA expression of iNOS, COX-2 and several proinflammatory cytokines, and rescue of DA neurons in the SN. Intriguingly, following treatment with pKr-2 in vitro, neurotoxicity was detected exclusively in co-cultures of mesencephalic neurons and microglia, but not microglia-free neuron-enriched mesencephalic cultures, indicating that microglia are required for pKr-2 neurotoxicity. Our results strongly suggest that microglia activated by endogenous compound(s), such as pKr-2, are implicated in the DA neuronal cell death in the SN.
authors.affiliation: Brain Disease Research Center, Ajou University School of Medicine, Suwon, Korea.
authors.forename: Sang Ryong
authors.initials: SR
authors.lastname: Kim
authors.forename: Eun Sook
authors.initials: ES
authors.lastname: Chung
authors.forename: Eugene
authors.initials: E
authors.lastname: Bok
authors.forename: Hyung Hwan
authors.initials: HH
authors.lastname: Baik
authors.forename: Young Cheul
authors.initials: YC
authors.lastname: Chung
authors.forename: So Yoon
authors.initials: SY
authors.lastname: Won
authors.forename: Eunhye
authors.initials: E
authors.lastname: Joe
authors.forename: Tae Hyong
authors.initials: TH
authors.lastname: Kim
authors.forename: Soung Soo
authors.initials: SS
authors.lastname: Kim
authors.forename: Min Young
authors.initials: MY
authors.lastname: Jin
authors.forename: Sang Ho
authors.initials: SH
authors.lastname: Choi
authors.forename: Byung Kwan
authors.initials: BK
authors.lastname: Jin
mesh_terms: D000818:Animals; D039481:CD11b Antigen / Q000032:analysis / Q000378:metabolism; D002478:Cells, Cultured; D018920:Coculture Techniques; D051546:Cyclooxygenase 2 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D052246:Cyclooxygenase 2 Inhibitors / Q000494:pharmacology; D004298:Dopamine / Q000378:metabolism*; D005260:Female; D005911:Gliosis / Q000139:chemically induced / Q000378:metabolism* / Q000503:physiopathology; D018836:Inflammation Mediators / Q000378:metabolism; D018082:Kringles / Q000502:physiology; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology; D017628:Microglia / Q000187:drug effects / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D010300:Parkinson Disease / Q000378:metabolism / Q000503:physiopathology; D011516:Prothrombin / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013378:Substantia Nigra / Q000187:drug effects / Q000378:metabolism* / Q000503:physiopathology
chemical_list: D039481:CD11b Antigen; D052246:Cyclooxygenase 2 Inhibitors; C509295:ITGAM protein, human; D018836:Inflammation Mediators; D012333:RNA, Messenger; D011516:Prothrombin; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D051546:Cyclooxygenase 2; D004298:Dopamine