| Summary: | 碩士 === 國立陽明大學 === 生命科學系暨基因體科學研究所 === 104 === Zn2+ is the second most abundant ion, next to iron, in the brain and is responsible for normal brain function. High concentration of Zn2+ is neurotoxic, and an imbalance of Zn2+ homeostasis has complex implication in brain function. These are three families of Zn2+-regulatory proteins: ZnT (SLC30 family), ZIP (SLC39 family), and cytosolic metallothioneins (MTs). They are responsible for the regulation of Zn2+ homeostasis and prevent the accumulation of excess intracellular free Zn2+ concentration ([Zn2+]i ). ZnT exports Zn2+ to extracellular space and organelles to decrease [Zn2+]i. ZIP imports Zn2+ to the cytosol to increase [Zn2+]i and MT interacts with Zn2+ at the thiol (-SH) group of the cysteine residues to regulate Zn2+ homeostasis. Dysregulation of Zn2+ homeostasis has been linked with Parkinson’s disease (PD). Elevated Zn2+ levels are found in the substantia nigra of PD patients. Previous studies show when dopamine (DA) and Zn2+ were infused into the striatum and substantia nigra, there is a synergistic effect on the depletion of striatal DA content in vivo. It is possible that DA and Zn2+ are involved in the degeneration of dopaminergic neurons. To understand the molecular mechanism of the synergistic effect of Zn2+ and DA, the role of Zn2+-regulatory proteins in the regulation of [Zn2+]i was studied using PC12 cell as a model system. PC12 cells express most ZnT, ZIP and MTs family proteins. FluoZin-3 is used as an indicator to monitor the dynamic changes of [Zn2+]i. Treatment of PC12 cells with Zn2+ alone increased [Zn2+]i, and in the presence of DA, [Zn2+]i was increased to a higher level. To examine whether oxidative stress generated from DA oxidation is responsible for the increase [Zn2+]i, H2O2 was used to induce oxidative stress. The results show that H2O2-induced a slower changes in [Zn2+]i with lower levels than that induced by DA. It is likely that oxidative stress can only partially account for the effects of DA. The effect of Zn2+ and DA on [Zn2+]i was inhibited by antioxidant including N-acetyl-L-cysteine, L-glutathione, ascorbic acid and uric acid. Next, we manipulated the only ZnT member in the plasma membrane, ZnT1 by overexpression or knockdown. We found the rate of Zn-induced [Zn2+]i rise was increased in ZnT1-knockdown cells and decreased in ZnT1-overexpressing cells. In the presence of DA, DA further enhanced the Zn2+-induced [Zn2+]i rise in ZnT1-knockdown cells. In contrast, the rate of Zn2+- or Zn2+/DA- induced [Zn2+]i was decreased in ZnT1-overexpressing cells. Our results suggest that ZnT1 is involved in the regulation of Zn2+/DA- induced [Zn2+]i rise in PC12 cells.
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