MPP+에 의한 신경독성에서 Oxidative stress와 caspase의 역할

Abstract

배양 5～7 일째 PC12 세포에서 MPTP나 MPP+에 의한 도파민성 신경독성이 유발되는 기전에 산화스트레스와 caspase 경로가 어떠한 작용을 하는지 밝히고자 Cu/Zn superoxide dismutase, melatonin, caspase 억제제, cyclosporin A를 사용하여 도파민성 신경세포독성에 대한 보호효과를 관찰하고자 하였다. MPP+에 의한 신경독성에 대하여 Cu/Zn superoxide dismutase와 melatonin의 여러 농도를 투여한 결과 superoxide dismutase는 MPP+에 의한 신경독성을 차단하지 못하였으나 100 nM melatonin은 신경독성에 대해 부분적인 보호효과가 있었다. MPTP에 의한 신경독성에 대하여 1 μM cyclosporin A와 caspase 억제제인 Z-VAD-fmk 100 μM를 처치한 결과 MPTP에 의한 신경독성은 차단하지 못하였다. 0.25～4 mM의 MPTP와 MPP+를 처리 한 후 caspase 3 활성의 유의한 변동은 확인 할 수 없었다. MPP+ 처리 시 caspase 3의 기질로 작용하는 PLC-γ1의 분절현상은 없었으나 MPP+ 농도에 의존적으로 PLC-γ1 발현은 감소하였다. 이상의 결과에서 MPP+가 도파민성 신경독성을 유발하는 기전으로 산소유리기의 생성이 중요하며, caspase 경로 이외의 세포자멸사 기전이 관여하는 것으로 생각된다. MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and its metabolite MPP+ (1-methyl-4-phenyl pyridium ion) are specific dopaminergic neurotoxins and induce parkisonism-like symptoms and signs in animal models. This study was designed to evaluate the roles of oxidative stress and caspase cascade in the intracellular mechanisms of dopaminergic neurotoxicity inducing necrosis and/or apoptosis. Nerve growth factor-differentiated PC12 cells were exposed to the dopaminergic neurotoxicity by adding 1 or 2 mM MPP+ or MPTP for 24 h, and assessed by viability, morphological observation, cell count and caspase 3 activity. Protective effects of several antioxidants and antiapoptotic agents on the dopaminergic neurotoxicity were evaluated. Cell viability, cell growth and generation of neurites markedly decreased in MPP+ or MPTP treated PC12 cells. Antioxidant, Cu/Zn superoxide dismutase (50～200 U/mL) did not improve the viability of the cells in MPP+ or MPTP-mediated neurotoxicity. However, MPP+-mediated viability was partially recovered by another antioxidant, melatonin (100 nM). Antiapoptotic agents, cyclosporin A (1 μM) and Z-VAD-fmk (100 μM) did not improve the viability of MPP+-mediated neurotoxicity. There were no significant changes in caspase 3 activity and PLC-γ1 expression in MPP+ or MPTP-mediated neurotoxicity. In conclusion, the mechanism causing MPP+-mediated dopaminergic neurotoxicity in PC12 cells seems to be partially related with oxidative stress, but not related with caspase 3 activation. On the other hand, the other apoptotic mechanism excluding caspase cascade may be an important role in MPP+-mediated neurotoxicity.