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Inhibition of Mitochondrial Clearance and Cu/Zn-SOD Activity Enhance 6-Hydroxydopamine-Induced Neuronal Apoptosis

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Abstract

Parkinson’s disease (PD) is a common movement disorder among neurodegenerative diseases, involving neuronal cell death in the substantia nigra of the midbrain. Although mechanisms of cell death in PD have been studied, the exact molecular pathogenesis is still unclear. Here, we explore the relationship between two types of cell death, autophagy and apoptosis, which have been studied separately in parkinsonian mimetic model of 6-hydroxydopamine (6-OHDA). 6-OHDA induced autophagy firstly and then later inhibition of autophagy flux occurred with apoptosis. The apoptosis was prevented by treatment of pan-caspase inhibitor, zVAD-fmk (benzyloxycarbonyl-VAD-fluoromethylketone (zVAD)), or early phase inhibitor of autophagy, 3-methyladenine (3-MA), indicating that autophagic induction was followed by the apoptosis. Interestingly, late step inhibitor of autophagy, bafilomycin A1 (BafA), aggravated 6-OHDA-induced apoptosis. This was associated with mitochondrial abnormality such as the inhibition of damaged mitochondrial clearance and aberrant increase of extracellular oxygen consumption. Furthermore, treatment of BafA did not inhibit 6-OHDA-mediated superoxide formation but strongly reduced the hydrogen peroxide production to below basal levels, indicating failure from superoxide to hydrogen peroxide. These results were accompanied by a lowered expression and activity of copper/zinc superoxide dismutase (Cu/Zn-SOD) but not of manganese SOD (MnSOD) and catalase. Thus, the present study suggests that crosstalk among apoptosis, autophagy, and oxidative stress is a causative factor of 6-OHDA-induced neuronal death and provides a mechanistic understanding of PD pathogenesis.

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Abbreviations

3-MA:

3-Methyladenine

6-OHDA:

6-Hydroxydopamine

AnV:

Annexin V

BafA:

Bafilomycin A1

COXIV:

Cytochrome C oxidase subunit IV

DCF-DA:

Dichlorodihydrofluorescein diacetate

DHE:

Dihydroethidium

DMSO:

Dimethylsulfoxide

FACS:

Fluorescence-activated cell sorting

GD:

Glucose deprivation

LC3:

Microtubule-associated protein 1A/1B-light chain 3

LDH:

Lactate dehydrogenase

MPTP:

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine

MTT:

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

PD:

Parkinson’s disease

PI:

Propidium iodide

ROS:

Reactive oxygen species

SOD:

Superoxide dismutase

tBID:

Truncated BH3-interacting domain death agonist

TNFa:

Tumor necrosis factor alpha

zVAD:

Benzyloxycarbonyl-VAD-fluoromethylketone

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Acknowledgments

We thank Dr. Daniel Principe for editing the language of the manuscript. This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2014R1A1A1002109). This research was also supported by the Hallym University Specialization Fund (HRF-S-51).

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The authors declare that they have no conflict of interests.

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    Authors and Affiliations

    1. Department of Anatomy and Neurobiology, College of Medicine, Hallym University, 1 Hallymdaehak-gil, Chuncheon, Gangwon-Do, 200-702, South Korea

      Sua In, Boyoung Choi, Bong-Geum Jang & Min-Ju Kim

    2. Department of Pharmacology, College of Medicine, Hallym University, Chuncheon, Gangwon-Do, 200-702, South Korea

      Chang-Won Hong

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    1. Sua In
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    Correspondence to Min-Ju Kim.

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    Sua In and Chang-Won Hong contributed equally to this work.

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    In, S., Hong, CW., Choi, B. et al. Inhibition of Mitochondrial Clearance and Cu/Zn-SOD Activity Enhance 6-Hydroxydopamine-Induced Neuronal Apoptosis. Mol Neurobiol 53, 777–791 (2016). https://doi.org/10.1007/s12035-014-9087-9

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