(I)Role of alphy-Synuclein in Regulation of Ca2+ Mobilization and Exocytosis; (II) The Effect of Squamocin on Mitochondrial Stability

• Main Authors: Hsiao-Yin Yang, 楊筱瑩
• Other Authors: Chung-Chih Lin
• Format: Others
• Language: en_US
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/15282611808231542496

碩士 === 國立陽明大學 === 生命科學暨基因體科學研究所 === 95 === (I) α-Synuclein is associated with several neurodegenerative diseases such as Parkinson’s disease (PD). α-Synuclein mainly localized in presynaptic terminals, has been reported to regulate the size of the presynaptic vesicular pool and vesicular turnover, synaptic plasticity and assemble of SNARE complex. The physiological role of α-synuclein in exocytosis is still unclear, especially whether α-synuclein regulate exocytosis via change of [Ca2+]i. Differentiated PC12 cells were transfected with NPY-Venus and ECFP-α-synuclein to examine whether α-synuclein affected exocytotic activity monitored by total internal reflection fluorescence microscopy (TIRFM). Upon ATP stimulation, the NPY-Venus fluorescence decay more rapidly in WT and A30PA53T α-synuclein overexpressing cells then control cells. More type 3 exocytotic events, i.e. the fluorescence decreased directly to background level upon stimulation, were observed in WT and mutant α-synuclein overexpressing cells. α-Synuclein may promote formation of SNARE complex and thus shorten the time for the exocytosis process of single exocytosis. Because Ca2+ triggers fusion of exocytosis, α-synuclein may affect calcium homeostasis to regulate exocytosis. Inhibitors of Ca2+ mobilization were applied to investigate the correlation among α-synuclein, Ca2+ mobilization and exocytosis. Overexpression of WT and A30PA53T α-synuclein reduced ATP-induced [Ca2+]i transient and Ca2+ decay after transient. The effects of overexpression of α-synuclein on [Ca2+]i and exocytosis upon ATP stimulation were diminished by application of thespsigargin (TG.) or u73122. TG treatment promoted Type 3 exocytosis in control and enhanced the exocytosis rate at later time in α-synuclein- overexpressing cells. It is possible that TG treatment may increase the available Ca2+ to trigger more exocytosis from slow releasable pool (SRP) and deplete total releasable pool at a faster rate. The results suggest that the effect of α-synuclein on exocytosis is via inhibition of TG-sensitive Ca2+ ER pool or pumps. (II) Abstract Annonaceous acetogenins, isolated from Annonaceae plants, have attracted considerable attention because of their special structure and wide range of biological activities, including parasiticidal, pesticidal, antimalavial and anti-tumor effects. Squamocin is a bis-tetrahydrofuran Annonaceous acetogenin, recently it has been shown to induce apoptosis in tumor cells. However, the subcellular target of squamocin is still unclear. The cytotoxicity of squamocin was measured by MTT assay, the ED50 of squamocin on CHO-K1 cells is about 8.2~16.4 μM. Since there are specific morphological changes in organelles during apoptosis, we focused on the change of morphology in nucleus, mitochondria, and ER to test whether apoptosis is due to apoptotic pathway. Squamocin treatment caused nucleus became smaller, condensed or horseshoe-like irregular morphology, mitochondrial fragmentation and ER reticulation. Squamocin induced significant mitochondrial fragmentation, release of cytochrome c to cytosol and lose of mitochondrial membrane potential. Apoptosis associated regulatory proteins, such as Bcl-2, Bax and Bak may contribute to apoptosis. Squamocin decreased the expression of Bcl-2, and promoted the Bax translcoated to mitochondria. Activation of Caspase is a key event in apoptosis. Caspase 8 inhibitor and caspase 9 inhibitor partially blocked the squamocin-induced mitochondrial fragmentation but can not rescue squamocin-induced cell death. Except squamocin induced mitochondrial fragmentation, there may be another mechanism for squamocin to result in cell death.