title: Proteasome inhibition induces selective motor neuron death in organotypic slice cultures.
abstract: A dysfunctional ubiquitin-proteasome system recently has been proposed to play a role in the pathogenesis of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). We have shown previously that spinal motor neurons are more vulnerable to proteasome inhibition-induced neurotoxicity, using a dissociated culture system. To confirm this toxicity, we used organotypic slice cultures from rat neonatal spinal cords, which conserve the structure of the spinal cord in a horizontal plane, enabling us to identify motor neurons more accurately than in dissociated cultures. Furthermore, such easy identifications make it possible to follow up the course of the degeneration of motor neurons. When a specific proteasome inhibitor, lactacystin (5 microM), was applied to slice cultures, proteasome activity of a whole slice was suppressed below 30% of control. Motor neurons were selectively damaged, especially in neurites, with the increase of phosphorylated neurofilaments. They were eventually lost in a dose-dependent manner (1 microM, P < 0.05; 5 microM, P < 0.01). The low capacity of Ca(2+) buffering is believed to be one of the factors of selectivity for damaged motor neurons in ALS. In our system, negative staining of Ca(2+)-binding proteins supported this notion. An intracellular Ca(2+) chelator, BAPTA-AM (10 microM), exerted a significant protective effect when it was applied with lactacystin simultaneously (P < 0.01). We postulate that proteasome inhibition is an excellent model for studying the mechanisms underlying selective motor neuron death and searching for new therapeutic strategies in the treatment of ALS.
authors.affiliation: Department of Neurology, Hokkaido University Graduate School of Medicine, Kita-ku, Sapporo, Japan. tujitti@jb3.so-net.ne.jp
authors.forename: Sachiko
authors.initials: S
authors.lastname: Tsuji
authors.forename: Seiji
authors.initials: S
authors.lastname: Kikuchi
authors.forename: Kazuyoshi
authors.initials: K
authors.lastname: Shinpo
authors.forename: Jun
authors.initials: J
authors.lastname: Tashiro
authors.forename: Riichiro
authors.initials: R
authors.lastname: Kishimoto
authors.forename: Ichiro
authors.initials: I
authors.lastname: Yabe
authors.forename: Shoichi
authors.initials: S
authors.lastname: Yamagishi
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Takeuchi
authors.forename: Hidenao
authors.initials: H
authors.lastname: Sasaki
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D002452:Cell Count / Q000379:methods; D016923:Cell Death / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002614:Chelating Agents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D007150:Immunohistochemistry / Q000379:methods; D009046:Motor Neurons / Q000166:cytology / Q000187:drug effects*; D016501:Neurites / Q000187:drug effects / Q000378:metabolism; D016900:Neurofilament Proteins / Q000145:classification / Q000378:metabolism; D009924:Organ Culture Techniques; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D051381:Rats; D017207:Rats, Sprague-Dawley; D013116:Spinal Cord / Q000166:cytology*; D013997:Time Factors
chemical_list: D002614:Chelating Agents; D016900:Neurofilament Proteins; D061988:Proteasome Inhibitors; C067713:lactacystin; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D046988:Proteasome Endopeptidase Complex; D000111:Acetylcysteine