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Pifithrin-Alpha Reduces Methamphetamine Neurotoxicity in Cultured Dopaminergic Neurons

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Abstract

Methamphetamine (Meth) is a widely abused stimulant. High-dose Meth induces degeneration of dopaminergic neurons through p53-mediated apoptosis. A recent study indicated that treatment with the p53 inhibitor, pifithrin-alpha (PFT-α), antagonized Meth-mediated behavioral deficits in mice. The mechanisms underpinning the protective action of PFT-α against Meth have not been identified, and hence, their investigation is the focus of this study. Primary dopaminergic neuronal cultures were prepared from rat embryonic ventral mesencephalic tissue. High-dose Meth challenge reduced tyrosine hydroxylase immunoreactivity and increased terminal deoxynucleotidyl transferase-mediated dNTP nick-end labeling (TUNEL) labeling. PFT-α significantly antagonized these responses. PFT-α also reduced Meth-activated translocation of p53 to the nucleus, an initial step before transcription. Previous studies have indicated that p53 can also activate cell death through transcription-independent pathways. We found that PFT-α attenuated endoplasmic reticulum (ER) stressor thapsigargin (Tg)-mediated loss of dopaminergic neurons. ER stress was further monitored through the release of Gaussia luciferase (GLuc) from SH-SY5Y cells overexpressing GLuc-based Secreted ER Calcium-Modulated Protein (GLuc-SERCaMP). Meth or Tg significantly increased GLuc release in to the media, with PFT-α significantly reducing GLuc release. Additionally, PFT-α significantly attenuated Meth-induced CHOP expression. In conclusion, our data indicate that PFT-α is neuroprotective against Meth-mediated neurodegeneration via transcription-dependent nuclear and -independent cytosolic ER stress pathways.

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Abbreviations

BSA:

Bovine serum albumin

CHOP:

CCAAT/enhancer-binding protein (C/EBP) homologous protein

DIV:

Day in vitro

DMEM:

Dulbecco’s modified Eagle medium

DMSO:

Dimethyl sulfoxide

ER:

Endoplasmic reticulum

GLuc:

Gaussia luciferase

Meth:

Methamphetamine

MPTP:

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

PBS:

Phosphate-buffered saline

PFA:

Paraformaldehyde

PFT-α:

Pifithrin-alpha

SERCaMP:

Secreted ER calcium-modulated protein

Tg:

Thapsigargin

TH:

Tyrosine hydroxylase

TUNEL:

Terminal deoxynucleotidyl transferase-mediated dNTP nick-end labeling

VM:

Ventral mesencephalon

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Funding

This research was supported in part by (i) the Ministry of Science and Technology, Taiwan (MOST 105-2320-B-400-012-MY3, 107-2314-B-030-009); (ii) the National Health Research Institutes, Taiwan; and (iii) the Intramural Research Programs of the National Institute on Aging and National Institute on Drug Abuse, NIH, USA.

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Authors and Affiliations

  1. Center for Neuropsychiatric Research, National Health Research Institutes, Zhunan, Taiwan

    Yun-Hsiang Chen, Eunkyung Bae, Hsi Chen, Seong-Jin Yu & Yun Wang

  2. Department of Life Science, Fu-Jen Catholic University, New Taipei City, Taiwan

    Yun-Hsiang Chen

  3. Molecular Mechanisms of Cellular Stress and Inflammation Unit, Intramural Research Program, National Institute on Drug Abuse, NIH, Baltimore, MD, USA

    Brandon K Harvey

  4. Translational Gerontology Branch, Intramural Research Program, National Institute of Aging, NIH, Baltimore, MD, USA

    Nigel H Greig

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  1. Yun-Hsiang Chen
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  2. Eunkyung Bae
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  3. Hsi Chen
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  4. Seong-Jin Yu
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  5. Brandon K Harvey
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  7. Yun Wang
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Correspondence to Yun Wang.

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Chen, YH., Bae, E., Chen, H. et al. Pifithrin-Alpha Reduces Methamphetamine Neurotoxicity in Cultured Dopaminergic Neurons. Neurotox Res 36, 347–356 (2019). https://doi.org/10.1007/s12640-019-00050-w

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