title: Proteomic identification of hippocampal proteins vulnerable to oxidative stress in excitotoxin-induced acute neuronal injury.
abstract: Excitotoxicity is involved in seizure-induced acute neuronal death, hypoxic-ischemic encephalopathy, and chronic neurodegenerative conditions such as Alzheimer's disease. Although oxidative stress has been implicated in excitotoxicity, the target proteins of oxidative damage during the course of excitotoxic cell death are still unclear. In the present study, we performed 2D-oxyblot analysis and mass spectrometric amino acid sequencing to identify proteins that were vulnerable to oxidative damage in the rat hippocampus during kainic acid (KA)-induced status epilepticus. We first investigated the time course in which oxidative protein damage occurred using immunohistochemistry. Carbonylated proteins, a manifestation of protein oxidation, were detected in hippocampal neurons as early as 3h after KA administration. Immunoreactivity for 8-hydroxy-2'-deoxyguanosine (8-OHdG) was also elevated at the same time point. The increase in oxidative damage to proteins and DNA occurred concomitantly with the early morphological changes in KA-treated rat hippocampus, i.e., changes in chromatin distribution and swelling of rough endoplasmic reticulum and mitochondria, which preceded the appearance of morphological features of neuronal death such as pyknotic nuclei and hypereosinophilic cytoplasm. Proteomic analysis revealed that several hippocampal proteins were consistently carbonylated at this time point, including heat shock 70kDa protein 4, valosin-containing protein, mitochondrial inner membrane protein (mitofilin), α-internexin, and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (14-3-3 protein). We propose that oxidative damage to these proteins may be one of the upstream events in the molecular pathway leading to excitotoxic cell death in KA-treated rat hippocampus, and these proteins may be targets of therapeutic intervention for seizure-induced neuronal death.
authors.affiliation: Department of Pathology, Institute for Developmental Research, Aichi Human Service Center, Aichi, Japan.
authors.forename: Ayako
authors.initials: A
authors.lastname: Furukawa
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Kawamoto
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Chiba
authors.forename: Shiro
authors.initials: S
authors.lastname: Takei
authors.forename: Sanae
authors.initials: S
authors.lastname: Hasegawa-Ishii
authors.forename: Noriko
authors.initials: N
authors.lastname: Kawamura
authors.forename: Keisuke
authors.initials: K
authors.lastname: Yoshikawa
authors.forename: Masanori
authors.initials: M
authors.lastname: Hosokawa
authors.forename: Shinji
authors.initials: S
authors.lastname: Oikawa
authors.forename: Masashi
authors.initials: M
authors.lastname: Kato
authors.forename: Atsuyoshi
authors.initials: A
authors.lastname: Shimada
mesh_terms: D000208:Acute Disease; D000818:Animals; D016923:Cell Death / Q000502:physiology; D004195:Disease Models, Animal; D018690:Excitatory Amino Acid Agonists / Q000633:toxicity; D006624:Hippocampus / Q000378:metabolism* / Q000473:pathology*; D007608:Kainic Acid / Q000633:toxicity; D008297:Male; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000378:metabolism / Q000473:pathology; D009498:Neurotoxins / Q000633:toxicity*; D018384:Oxidative Stress / Q000502:physiology*; D040901:Proteomics / Q000379:methods*; D051381:Rats; D017208:Rats, Wistar; D013226:Status Epilepticus / Q000378:metabolism* / Q000473:pathology*
chemical_list: D018690:Excitatory Amino Acid Agonists; D009419:Nerve Tissue Proteins; D009498:Neurotoxins; D007608:Kainic Acid