Selective Cytotoxic Mechanism of Homocysteine to Motor Neuronal Cells Expressing the Mutant Cu,Zn-superoxide Dismutase

J Korean Neurol Assoc > Volume 24(1); 2006 > Article

Journal of the Korean Neurological Association 2006;24(1): 66-75.

돌연변이 Cu,Zn-superoxide dismutase 유전자를 발현하는 운동신경세포에 대한 Homocysteine의 선택적 세포독성 기전

성정준, 김현정 김성민 민주홍 홍윤호b 박경석 김만호 이석호a 호원경a 이광우

서울대학교 의과대학 신경과학교실, 생리학교실a, 서울대학교 보라매병원 신경과b

Selective Cytotoxic Mechanism of Homocysteine to Motor Neuronal Cells Expressing the Mutant Cu,Zn-superoxide Dismutase

Jung-Joon Sung

Departments of Neurology and Physiologya, College of Medicine, Seoul National University, Seoul; Department of Neurology, Seoul National University Boramae Hospitalb, Seoul, Korea

Abstract

Background: Mutations in Cu, Zn-superoxide dismutase (SOD1) cause about 20% of familial amyotrophic lateral sclerosis (FALS) cases. The mechanism of late-onset disease manifestation despite the innate mutation has no clear explanation. The relationship between homocysteine (HC) and amyotrophic lateral sclerosis (ALS) has not been investigated fully, in spite of the similarity in their pathogenesis.
Methods: We investigated the effect of HC on the motor neuronal cell-line (VSC4.1) transfected with SOD1 of either wild-type or mutant forms (G93A and A4V) using various methods including the MTT assay for the cytotoxic assay, the immunocytochemical staining using anti-SOD1 for the aggregation of SOD1, the western blotting using anti-nitrotyrosine and anti-DNPH for the oxidative protein damage, and the measurement of the intracellular Ca2+ concentration using Fura2-AM.
Results: In the MTT assay, the HC induced significant cytotoxicity in the mutants, as compared with wild-type. This HC-induced cytotoxicity was inhibited by the trolox and the bathocuproinedisulfonate (BC). HC increased the carbonylation and nitrosylation of the mutant proteins. HC also increased significant SOD1-aggregation in mutants. This HC-induced SOD1-aggregation in mutants was inhibited by trolox, N-nitro-L-arginine methyl ester, BC, and z-VAD-FMK. HC did not change the intracellular concentration of Ca2+ in the mutants compared with the wild-type.
Conclusions: The authors showed that the vulnerability of the SOD1 mutant motor neuronal cells to HC involves the copper-mediated oxygen radical toxicity, and that HC may be a lifelong precipitating factor in some forms of FALS, suggesting a possible treatment modality with vitamin supplements.