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  4. The Roles of Intracellular Zinc Elevation in the Dopamine-induced Autophagy Activation in PC12 Cells and Cultured Rat Embryonic Cortical Neurons
 
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The Roles of Intracellular Zinc Elevation in the Dopamine-induced Autophagy Activation in PC12 Cells and Cultured Rat Embryonic Cortical Neurons

Date Issued
2016
Date
2016
Author(s)
Hung, Hui-Hsing
URI
http://ntur.lib.ntu.edu.tw//handle/246246/272432
Abstract
Zinc ion (Zn2+), one of the most abundant trace metals in the brain, is essential for neuronal activities but induces toxicity when the concentration is abnormally high. Dopamine is an important neurotransmitter and is involved in autophagy, apoptosis, and neuromodulation. Autophagy plays an important dual role in neuronal cell death and cell survival. However, the mechanism behind the toxic effects of dopamine in neurodegenerative diseases is not clear. In this report, we first characterized how the high concentrations of dopamine and Zn2+ induced autophagy in PC12 cells and cultured embryonic cortical neurons; then investigated how dopamine elevated the intracellular Zn2+ concentration ([Zn2+]i) for autophagy activation. High concentrations of dopamine and Zn2+ increased the number and size of the aggregates of EGFP-LC3 expressed in cells as an indicator of autophagosome formation. The Western blot analysis showed the lipidation level of LC3 increased by these treatments in PC12 cells and cultured neurons. Introducing siRNA against ATG7, an initiator protein of autophagy, and blocking the phosphatidylinositol 3-phosphate kinase inhibited the formation of EGFP-LC3 aggregates in PC12 cells. In addition, blocking autophagosome formation increased the level of phosphatidylserine exposure on the outer membrane leaflet in PC12 cells when treated with dopamine or Zn2+. Dopamine or Zn2+ treatment significantly elevated [Zn2+]i; however, pretreatment of PC12 cells and neurons with a Zn2+ chelator suppressed the dopamine-induced autophagosome formation and LC3 lipidation. In neurons, inhibitors against the dopamine D1-like receptor, cAMP-dependent protein kinase (PKA), and NO synthase suppressed the dopamine-induced [Zn2+]i elevation. PKA activators and NO generators directly elevated the [Zn2+]i in cultured neurons. Using cell fractionation, proteins with M.W. values between 5 and 10 kDa were found to release Zn2+ following NO stimulation. In addition, chelating the [Zn2+]i elevation blocked the NO-activated autophagy. Therefore, our results indicate that treating cells with dopamine and Zn2+ results in the activation of the autophagy pathway in an effort to enhance cell survival. Furthermore, we demonstrate that the dopamine-induced [Zn2+]i elevation is mediated by the D1-like receptor-PKA-NO pathway and is crucial for autophagy activation.
Subjects
Zn2+
dopamine
autophagy
NO
neuron
Type
thesis
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