Prothrombin kringle-2 induces death of mesencephalic dopaminergic neurons in vivo and in vitro via microglial activation.
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.
Kim
Sang Ryong
SR
Brain Disease Research Center, Ajou University School of Medicine, Suwon, Korea.
Chung
Eun Sook
ES
Bok
Eugene
E
Baik
Hyung Hwan
HH
Chung
Young Cheul
YC
Won
So Yoon
SY
Joe
Eunhye
E
Kim
Tae Hyong
TH
Kim
Soung Soo
SS
Jin
Min Young
MY
Choi
Sang Ho
SH
Jin
Byung Kwan
BK
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
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