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Quercetin Exerts Differential Neuroprotective Effects Against H2O2 and Aβ Aggregates in Hippocampal Neurons: the Role of Mitochondria

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Abstract

Amyloid-β peptide (Aβ) is one of the major players in the pathogenesis of Alzheimer’s disease (AD). Despite numerous studies, the mechanisms by which Aβ induces neurodegeneration are not completely understood. Oxidative stress is considered a major contributor to the pathogenesis of AD, and accumulating evidence indicates that high levels of reactive oxygen species (ROS) are involved in Aβ-induced neurodegeneration. Moreover, Aβ can induce the deregulation of calcium homeostasis, which also affects mitochondrial function and triggers neuronal cell death. In the present study, we analyzed the effects of quercetin, a plant flavonoid with antioxidant properties, on oxidative stress- and Aβ-induced degeneration. Our results indicate that quercetin efficiently protected against H2O2-induced neuronal toxicity; however, this protection was only partial in rat hippocampal neurons that were treated with Aβ. Treatment with quercetin decreased ROS levels, recovered the normal morphology of mitochondria, and prevented mitochondrial dysfunction in neurons that were treated with H2O2. By contrast, quercetin treatment partially rescued hippocampal neurons from Aβ-induced mitochondrial injury. Most importantly, quercetin treatment prevented the toxic effects that are induced by H2O2 in hippocampal neurons and, to a lesser extent, the Aβ-induced toxicity that is associated with the superoxide anion, which is a precursor of ROS production in mitochondria. Collectively, these results indicate that quercetin exerts differential effects on the prevention of H2O2- and Aβ-induced neurotoxicity in hippocampal neurons and may be a powerful tool for dissecting the molecular mechanisms underlying Aβ neurotoxicity.

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Abbreviations

Aβ:

Amyloid-β peptide

Aβo:

Amyloid-β peptide oligomers

AD:

Alzheimer’s disease

ARE:

Antioxidant response element

AraC:

1-β-d-Arabinofuranosylcytosine

ATP:

Adenosine triphosphate

CypD:

Cyclophilin D

DMSO:

Dimethyl sulfoxide

KRH:

Krebs-Ringer-HEPES

LTP:

Long-term potentiation

MAP:

kinase Mitogen-activated protein kinase

MTT:

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

mtDNA:

Mitochondrial DNA

MnSOD:

Superoxide dismutase 2, mitochondrial

NAD:

Nicotinamide adenine dinucleotide

Nrf2:

Nuclear factor erythroid 2-related factor 2

NOS:

Nitric oxide synthase

PON2:

Paraoxonase 2

PCG-1α:

Peroxisome proliferator-activated receptor gamma coactivator 1-alpha

PKC:

Protein kinase C

PD:

Parkinson’s disease

PBS:

Phosphate-buffered saline

SIRT1:

NAD-dependent deacetylase sirtuin-1

TMRM:

Tetramethylrhodamine, methyl ester

ROS:

Reactive oxygen species

ROI:

Region of interest

2,7-DCF:

2′,7′-Dichlorofluorescein

H2O2 :

Hydrogen peroxide

fEPSP:

Field excitatory postsynaptic potential

β-Tubulin-III:

β-Tubulin isotype III

GFAP:

Glial fibrillary acidic protein

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Acknowledgments

This work was supported by grants from the Basal Centre for Excellence in Science and Technology (Conicyt-PFB 12/2007, CARE UC), Fondecyt No. 1160724 to N.C.I, Fondecyt No. 11121206 to W.C., and Fondecyt No. 1140968 to R.A.Q; research team project in Science and Technology (ACT1411) to WC and R.A.Q; and predoctoral fellowship to FJC from CONICYT. JAG is a Ph.D. student for Universitat Pompeu Fabra, Barcelona, Spain, and CBL is a student of a Master (MSc) Program in Biochemistry, University of Chile, Chile.

Author Contributions

JAG designed and coordinated the study as well as conducted most of the neuronal culture and experiments; he also drafted the manuscript. CBL assisted in certain experimental procedures, performed some of the statistical analyses, and edited the manuscript. RAQ performed the neuronal cultures and membrane mitochondrial potential measurements and edited the manuscript. FJC prepared and assisted in the electrophysiological experiments, aided with transgenic animal development, and edited the manuscript. WC assisted with the multiplex analysis for experiments and with revising the manuscript. NCI conceived of the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.

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Correspondence to Waldo Cerpa or Nibaldo C. Inestrosa.

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Electronic Supplementary Material

Supplementary Fig. 1.

Hippocampal neurons cultures challenged with Aβo. Hippocampal neurons were cultured for 15 DIV, with neurobasal/B27, at 37ºC, on coverslips (12mm), treated with AraC for 36 hours, at day 4 of cultures to remove proliferating cells. Cultures were depleted of supplements and antibiotics and the cells were maintained in neurobasal, until the experiments were performed and used for different assays with quercetin. A. Neurons were pretreated with quercetin for 30 min, then challenged with 10nM Aβo for 2 hr, stain with antibodies against MAP1B to detect neurons and TOM20 to detect mitochondria, photographed on Carl Zeiss LSM confocal microscope at different magnifications (10x, 20x and 63x), a 63x oil objective were obtained a zoom micrographs to detect mitochondrial morphology (white square: a,b: white arrow). B. Sister cultures were stained with β-tubulin III (red label) to detect neurons and GFAP (green label) to detect non-neuronal cells. (GIF 2542 kb)

High resolution image (TIFF 5357 kb)

Supplementary Fig. 2.

Hippocampal neurons cultures challenged with H2O2. Cultures were depleted of supplements and antibiotics and the cells were maintained in neurobasal, until the experiments were performed and used for different assays with quercetin and H2O2. A. Neurons were pretreated with quercetin for 30 min, then challenged with 10nM H2O2 for 2 hr, stain with antibodies against MAP1B to detect neurons and TOM20 to detect mitochondria, photographed on Carl Zeiss LSM confocal microscope at different magnifications (10x, 20x and 63x), a 63x oil objective were obtained a zoom micrographs to detect mitochondrial morphology (white square: a,b: white arrow). B. Sister cultures were stained with β-tubulin III (red label) to detect neurons and GFAP (green label) to detect non-neuronal cells. (GIF 2451 kb)

High resolution image (TIFF 5434 kb)

Supplementary Fig. 3.

Oxidative stress marker and behavioral impairment are increase in CA1 region of SOD2+/- animals. A. Representative pictures of immunofluorescence again 8-hidroxiguanine in the CA1 region of hippocampal slices from WT and SOD2+/- mice at 2 and 5 month old (a). (bar, 100 μm); original magnification, x400. Quantification of intensity stain in CA1 region. (b). B. Analysis of swimming path to reach the hidden platform in the Morris wáter maze test of the WT and SOD2+/- animals (a). Escape latency (time to reach the hidden platform) of WT and SOD2+/- animals on day 1 and day 9 (b). Representative swimming strategies on day 9 of WT and SOD2+/- at different ages (c). Three animals were used per immunoflurescence analysis, and 6 animals were used for behavioral test per experimental group. Data are means ± S.E. Statistical differences were calculated by ANOVA, followed by post hoc Bonferroni’s test. Asterisks indicate statistical significance if the observed differences (*p>0.05; **p>0.01;***p>0.001). (GIF 426 kb)

High resolution image (TIFF 641 kb)

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Godoy, J.A., Lindsay, C.B., Quintanilla, R.A. et al. Quercetin Exerts Differential Neuroprotective Effects Against H2O2 and Aβ Aggregates in Hippocampal Neurons: the Role of Mitochondria. Mol Neurobiol 54, 7116–7128 (2017). https://doi.org/10.1007/s12035-016-0203-x

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