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Dopamine- and zinc-induced autophagosome formation facilitates PC12 cell survival

Cell Biology and Toxicology volume 29, pages 415–429 (2013)Cite this article

Abstract

Dopamine oxidation and divalent cations have been reported to induce neuronal cell death. Although autophagy is involved in neuronal cell death, it has also been suggested to facilitate cell survival. We sought to investigate the role of autophagy in PC12 cells and cultured neurons treated with dopamine and Zn2+. Cells expressing EGFP-LC3 were treated with high concentrations of dopamine and Zn2+, and the formation of EGFP-LC3 fluorescence aggregates was monitored. Our results showed a significant increase in the number of fluorescent puncta in the cytosol of PC12 cells treated with these chemicals. These treatments enhanced LC3 lipidation levels in PC12 cells. Decreasing the ATG7 protein level using specific small interference RNA (siRNA) and pretreating with phosphatidylinositol 3-phosphate kinase blockers, wortmannin and LY294002, inhibited puncta formation. Dopamine or Zn2+ treatment significantly elevated the intracellular Zn2+ concentration ([Zn2+] i ); however, inhibiting the [Zn2+] i elevation in dopamine-treated cells suppressed the puncta formation. LY294002 or siRNA-directed members of the autophagy pathway increased the fraction of phosphatidylserine present on the outer membrane leaflet in PC12 cells treated with dopamine or Zn2+, suggesting an increase in apoptosis. Primary embryonic midbrain neurons expressing EGFP-LC3 also displayed a significant increase in the number of fluorescent aggregates in cells upon treatment with dopamine or Zn2+. Dopamine or Zn2+ treatment significantly elevated the [Zn2+] i in neurons and caused neuronal death. 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.

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Abbreviations

Annexin V-FITC:

Annexin V-fluorescein isothiocyanate

Atg8-PE:

Atg8-phosphatidylethanolamine

BafA1:

Bafilomycin A1

DAPI:

4′-6-Diamidino-2-phenylindole

HBSS:

Hank’s buffered salt solution

MTT:

3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

PAS:

Pre-autophagosomal structure

PBS:

Phosphate-buffered saline

PD:

Parkinson’s disease

PI:

Propidium iodide

PI3K:

Phosphatidylinositol 3-phosphate kinase

PS:

Phosphatidylserine

TPEN:

N,N,N′,N′-tetrakis-(2-pyridylmethyl)-thylenediamine

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Acknowledgments

We would like to thank Professor Chun-Jen Chen for his help with the flow cytometry, Professor Hsin-Yu Lee for his help in MTT assay, and Technology Commons, College of Life Science, National Taiwan University for their help in the confocal microscopy. We thank Mr. Chih-Chen Yang and Dr. Yung-Feng Liao (Academia Sinica, Taipei, Taiwan) for their help with the PC12 cell cultures and our time-course experiment. This study was supported by the National Science Council of Taiwan (http://web1.nsc.gov.tw/mp.aspx?mp=7) under grant nos. NSC-99-2628-B-002-054-MY3 and NSC-100-2627-M-002-013.

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Authors and Affiliations

  1. Institute of Zoology, National Taiwan University, 1 Sec. 4, Roosevelt Road, Taipei, 106, Taiwan

    Hui-Hsing Hung, Wei-Pang Huang & Chien-Yuan Pan

  2. Department of Life Science, National Taiwan University, 1 Sec. 4, Roosevelt Road, Taipei, 106, Taiwan

    Wei-Pang Huang & Chien-Yuan Pan

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  1. Hui-Hsing Hung
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  2. Wei-Pang Huang
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Correspondence to Chien-Yuan Pan.

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Hung, HH., Huang, WP. & Pan, CY. Dopamine- and zinc-induced autophagosome formation facilitates PC12 cell survival. Cell Biol Toxicol 29, 415–429 (2013). https://doi.org/10.1007/s10565-013-9261-2

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  • DOI: https://doi.org/10.1007/s10565-013-9261-2

Keywords

  • Apoptosis
  • Autophagosome
  • Dopamine
  • Intracellular Zn2+ concentration
  • Phosphatidylserine
  • Zinc