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Toxicity of 6-hydroxydopamine: live cell imaging of cytoplasmic redox flux

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Abstract

Oxidative stress contributes to several debilitating neurodegenerative diseases. To facilitate direct monitoring of the cytoplasmic oxidation state in neuronal cells, we have developed roTurbo by including several mutations: F223R, A206K, and six of the mutations for superfolder green fluorescent protein. Thus we have generated an improved redox sensor that is much brighter in cells and oxidizes more readily than roGFP2. Cytoplasmic expression of the sensor demonstrated the temporal pattern of 6-hydroxydopamine (6-OHDA) induced oxidative stress in a neuroblastoma cell line (SH-SY5Y). Two distinct oxidation responses were identified in SH-SY5Y cells but a single response observed in cells lacking monoamine transporters (HEK293). While both cell lines exhibited a rapid transient oxidation in response to 6-OHDA, a second oxidative response coincident with cell death was observed only in SH-SY5Y cells, indicating an intracellular metabolism of 6-OHDA, and or its metabolites are involved. In contrast, exogenously applied hydrogen peroxide induced a cellular oxidative response similar to the first oxidation peak, and cell loss was minimal. Glucose deprivation enhanced the oxidative stress induced by 6-OHDA, confirming the pivotal role played by glucose in maintaining a reduced cytoplasmic environment. While these studies support previous findings that catecholamine auto-oxidation products cause oxidative stress, our findings also support studies indicating 6-OHDA induces lethal oxidative stress responses unrelated to production of hydrogen peroxide. Finally, temporal imaging revealed the sporadic nature of the toxicity induced by 6-OHDA in neuroblastoma cells.

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Abbreviations

DTT:

Dithiothreitol

GFP:

Green fluorescent protein

GSH/GSSG:

Glutathione/glutathione-disulfide ratio

NAC:

N-acetyl cysteine

6-OHDA:

6-Hydroxydopamine

PPP:

Pentose phosphate pathway

RNS:

Reactive nitrogen species

ROS:

Reactive oxygen species

RLU:

Relative luminescent units

roGFP:

Redox sensitive green fluorescent protein

SEM:

Standard error of the mean

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Acknowledgments

We thank to Michael Hodge for technical assistance and Dr. Ramanjaneya Mula for critical assessment of the manuscript. This work was supported by grants from the Arthritis & Chronic Pain Research Institute and the Alzheimer’s & Aging Research Center and was supported by the State of Florida, Executive Office of the Governor’s Office of Tourism, Trade, and Economic Development.

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Authors and Affiliations

  1. Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway, Port St. Lucie, FL, 34987, USA

    Colette T. Dooley, Ling Li, Jaime A. Misler & Jane H. Thompson

Authors
  1. Colette T. Dooley
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  2. Ling Li
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  3. Jaime A. Misler
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Corresponding author

Correspondence to Colette T. Dooley.

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Supp Fig. 1

Sequence map for mutations included in roTurbo (JPEG 79 kb)

High-resolution image (JPEG 102 kb)

Supp Fig. 2

Formation of auto-oxidation products of 6-OHDA in DMEM. a Auto-oxidation of 6-OHDA was measured spectrophotometrically, absorption of p-quinone was monitored at 490 nm. A stock solution of 6-OHDA (40 mM) was prepared in 100% ethanol. The experiment was initiated by the addition of 6-OHDA (100 μM final) to DMEM/F12/10% FBS (phenol-red-free). The reaction was maintained at 37oC, and absorbance was read every 10 s. Oxidation was monitored with or without NAC (10 mM). b Hydrogen peroxide formation by 6-OHDA was measured by Fluoro H2O TM2 (Cell Technology Inc, Mountain View, CA) according to manufacturer’s protocol. The stock solution of 6-OHDA was diluted in DMEM/F12 with 10% FBS (phenol-red-free) to give a final concentration of 100 μM. Following incubated for 2, 4, 6, 8, or 10 min, 5 μl of the reaction mixture was added to 45 μl HBSS followed by 50-μl reaction cocktail (50 μM detection reagent and 10 U/ml hydrogen peroxidase) and the reaction incubated at room temperature for 10 min. The amount of fluorescent product, resorufin, was measure at Ex 570 nm and Em 600 nm. A standard curve was prepared from 20 mM H2O2 diluted in the same buffer (5 ul DMEM/F12/10% FBS + 45 ul HBSS) and used to determine the concentrations of H2O2 generated by 6-OHDA auto-oxidation (JPEG 45 kb)

High-resolution image (TIFF 688 kb)

Supp Fig. 3

Control experiments for SH-SY5Y cells. a Cells incubated with media. b Cells incubated with media lacking glucose cells did not exhibit signs of oxidative stress as monitored by roTurbo (upper panel) and cells numbers remained constant throughout a 12-h period (lower panel). c Cells incubated with both media containing lowered concentrations of glucose, and 10 mM N-acetyl cysteine did not exhibit any signs of oxidative stress as monitored by roTurbo (upper panel) and cells numbers remained constant throughout a 12-h period (lower panel). Points represent the averages from two to four experiments. d H2O2 causes cell toxicity in HEK293 cells beyond the incubation period used in this study. Error bars represent SEM (JPEG 85 kb)

High-resolution image (TIFF 2179 kb)

Supp Fig. 4a

LC-MS mass spectrometry in DMEM or H2O. Incubation of 6-OHDA (100 μM) and N-acetyl cysteine (1 mM) in water for 2 h. (JPEG 100 kb)

High-resolution image (TIFF 1685 kb)

Supp Fig. 4b

LC-MS mass spectrometry in DMEM or H2O. Incubation of 6-OHDA (400 μM) and N-acetyl cysteine (10 mM) in water for 24 h. (JPEG 86 kb)

High-resolution image (TIFF 1685 kb)

Supp Fig. 4c

LC-MS mass spectrometry in DMEM or H2O. Upper panel: Incubation of 6-OHDA (400 μM) in DMEM for 6 h. Lower panel: incubation of 6-OHDA (400 μM) and N-acetyl cysteine (1 mM) in DMEM for 7 h (JPEG 88 kb)

High-resolution image (TIFF 1504 kb)

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Dooley, C.T., Li, L., Misler, J.A. et al. Toxicity of 6-hydroxydopamine: live cell imaging of cytoplasmic redox flux. Cell Biol Toxicol 28, 89–101 (2012). https://doi.org/10.1007/s10565-011-9209-3

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  • DOI: https://doi.org/10.1007/s10565-011-9209-3

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