Resveratrol Ameliorates Tau Hyperphosphorylation at Ser396 Site

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Resveratrol ameliorates tau hyperphosphorylation at Ser396 site and oxidative damage in rat hippocampal slices exposed to vanadate: Implication of ERK1/2 and GSK-3# signaling cascades Kyoung A Jhang, Jin-Sun Park, Hee-Sun Kim, and Young Hae Chong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03252 • Publication Date (Web): 12 Oct 2017 Downloaded from http://pubs.acs.org on October 15, 2017

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Journal of Agricultural and Food Chemistry

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Title: Resveratrol ameliorates tau hyperphosphorylation at Ser396 site and

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oxidative damage in rat hippocampal slices exposed to vanadate: Implication of

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ERK1/2 and GSK-3β signaling cascades

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Authors: Kyoung A Jhang†, Jin-Sun Park‡, Hee-Sun Kim*‡and Young Hae Chong*†

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†

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Affiliation:

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Neuroscience, Ewha Medical Research Institute, School of Medicine, Ewha Womans

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University, Seoul, Republic of Korea; ‡Department of Molecular Medicine, Tissue Injury

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Defense Research Center, School of Medicine, Ewha Womans University, Seoul, Republic of

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Korea.

Department of Microbiology and Division of Molecular Biology and

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* Correspondence to: Young Hae Chong, PhD, Department of Microbiology, School of

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Medicine, Ewha Womans University, 1071 Anyangchun-ro, Yangchun-Gu, Seoul 07985,

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Republic

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[email protected] or Hee-Sun Kim, PhD, Department of Molecular Medicine, Ewha

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Womans University, School of Medicine, 1071 Anyangchun-ro, Yangchun-Gu, Seoul 07985,

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Republic of Korea, Tel.: 82-2-2650-5823, Fax: 82-2-2653-8891 Email: [email protected]

of

Korea,

Tel.:82-2-2650-5739,

Fax:82-2-2653-8891,

23

1

ACS Paragon Plus Environment

E-mail:


24 25

ABSTRACT

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The objective of this study was to investigate the effect of resveratrol (a natural polyphenolic

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phytostilbene) on tau hyperphosphorylation and oxidative damage induced by sodium

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orthovanadate (Na3VO4), the prevalent species of vanadium (vanadate), in rat hippocampal

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slices. Our results showed that resveratrol significantly inhibited Na3VO4-induced

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hyperphosphorylation of tau at Ser396 (p-S396-tau) site, which is upregulated in the

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hippocampus of Alzheimer's disease (AD) brains and principally linked to AD-associated

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cognitive dysfunction. Subsequent mechanistic studies revealed that reduction of ERK1/2

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activation was involved in the inhibitory effect of resveratrol by inhibiting the ERK1/2

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pathway with SL327 mimicking the aforementioned effect of resveratrol. Moreover,

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resveratrol potently induced GSK-3β Ser9 phosphorylation and reduced Na3VO4-induced p-

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S396-tau levels, which were markedly replicated by pharmacologic inhibition of GSK-3β

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with LiCl. These results indicate that resveratrol could suppress Na3VO4-induced p-S396-tau

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levels via downregulating ERK1/2 and GSK-3β signaling cascades in rat hippocampal slices.

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In addition, resveratrol diminished the increased extracellular reactive oxygen species

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generation and hippocampal toxicity upon long-term exposure to Na3VO4 or FeCl2. Our

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findings strongly support the notion that resveratrol may serve as a potential nutraceutical

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agent for AD.

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Keywords: AD: Resveratrol, Alzheimer’s disease, vanadate, p-S396-tau, oxidative damage

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Introduction

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Alzheimer’s disease (AD) is the most common neurodegenerative disorder primarily

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affecting the elderly population over the age of 60 years. Most AD cases are sporadic where

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the origin of the disease is not known but might be influenced by multiple factors including

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environmental exposure, genetic risk factors, and age. The number of patients suffering from

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AD is expected to triple by 2050 if no preventative measures are developed.1 The major

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pathological changes of AD are amyloid-β (Aβ) deposition, neurofibrillary tangles (NFTs)

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formation, oxidative damage, and neuroinflammation that, in concert, lead to neocortical and

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hippocampal atrophy, memory dysfunction and decline of cognition in AD patients.2,3

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Clinical evidence indicates that the number of NFTs correlates with disease progression and

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the severity of dementia in AD patients than other changes. Recent studies have also shown

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that oxidative stress can promote AD progression.4-6 To date, available therapeutic agents are

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only able to slow the progression of this devastating dementia, with limited benefits.7 Owing

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to the heterogeneity of AD, pharmacotherapies targeting multiple levels of AD pathology are

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needed to alleviate cognitive dysfunction.8,9

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NFTs are mainly composed of hyperphosphorylated microtubule-associated protein tau in

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the brains of AD patients. While the mechanisms leading to the formation of NFTs are still

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elusive, an imbalance between protein kinases and protein phosphatases has been well

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recognized as the direct cause for abnormal tau hyperphosphorylation and synapse

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dysfunction in AD pathology.10 Indeed, tau kinases are upregulated in brains from AD

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patients, while the expression levels of tau phosphatases are reduced.11 Earlier studies have

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demonstrated diffuse phosphorylated tau protein at Ser396 site (p-S396-tau) in senile plaques

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(SPs) and NFTs in the brains of human AD patients,12 postulating that the increased levels p-

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S396-tau might be a reliable marker of AD severity. In addition, p-S396-tau protein has been 3



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found in early phospho-tau aggregates with well-preserved neuronal soma, validating p-

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S396-tau as an efficient marker for AD cytopathology following the progression of tau

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aggregation into NFTs.13 Moreover, elevated plasma levels of p-S396-tau contained within

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neuron-derived exosomes (NDEs) traffic from the central nervous system (CNS) to blood

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have allowed accurate prediction of conversion from mild cognitive impairment (MCI) to AD

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dementia.14 This group also demonstrated that plasma NDEs from demented patients seeded

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tau aggregation in hippocampus and induced AD-like neuropathology in normal mouse CNS

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mice. These findings altogether implicate that abnormal tau phosphorylation at Ser396 site is

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an early key step in the development of NFT pathology in AD brains, postulating p-S396-tau

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as a diagnostic biomarker and a potential target for delaying progression of MCI to AD

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dementia.

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Among

naturally

occurring

dietary

phytochemicals,

resveratrol

(trans-3,4′,5-

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trihydroxylstilbene), a phytoalexin with a stilbene structure present in red wine, grapes,

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berries, chocolate and peanuts, has received considerable attention as an alternative candidate

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for AD therapy15,16 due to its anti-oxidant, anti-inflammatory, anti-aging and neuroprotective

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effects. Despite of low water solubility, resveratrol can show blood–brain barrier (BBB)

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penetration ability that elicits its effects in the brain.17,18 Compelling evidence has shown that

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the phytochemical resveratrol facilitates non-amyloidogenic breakdown of the amyloid

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precursor protein (APP), and promotes removal of neurotoxic Aβ peptides, a critical step in

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preventing and slowing down AD pathology.15,16 Indeed, resveratrol has been shown to

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disrupt preformed Aβ aggregates, and diminish plaque formation in Tg19959 mice containing

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APP695 with two familial AD mutations (KM670/671NL and V717F).19 Moreover,

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resveratrol can prevent memory decline and markedly decrease oxidant stress indices in the

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ovariectomized rats chronically treated with D-galactose.20 These findings strongly suggest 4


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that resveratrol as a potent drug can target multiple levels of the underlying pathology of AD

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through several mechanisms of action by which resveratrol exhibits its modulation of tau

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hyperphosphorylation at Ser396 site are unclear.

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In addition to its potential inhibition of Na+/K+ adenosine triphosphatase (ATPases),

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vanadate is the broad-spectrum phosphatase inhibitor that can specifically inhibit protein

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phosphatases 2A (PP2A) and protein tyrosine phosphatase 1B (PTP1B), key Tau

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phosphatases implicated in AD.21,22 Importantly, earlier studies reported that vanadium

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compound might induce hyperphosphorylation of tau23 and oxidative stress24, thus result in

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AD-like damage. However, few studies try to understand how vanadate affects

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hyperphosphorylation of tau at Ser396 site and oxidative stress, which are principally linked

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to AD-associated cognitive dysfunction, and how resveratrol modulate this process at the

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molecular level.

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In this regard, we sought to determine the effect of resveratrol on two major pathological

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features of AD: the abnormal hyperphosphorylation of tau protein and oxidative damage in

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rat hippocampal slices after exposure to the prevalent species of vanadium, sodium

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orthovanadate (Na3VO4). Furthermore, the underlying signaling pathways for these effects

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were studied. Subsequent mechanistic studies revealed that resveratrol potently suppressed

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Na3VO4-induced hyperphosphorylation of tau at Ser396 site via downregulating ERK1/2 and

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GSK-3β signaling cascades. Moreover, resveratrol rescued hippocampal slices from Na3VO4-

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induced oxidative damage via reducing extracellular ROS levels independent of ERK1/2 and

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GSK-3β signaling cascades. Thus, resveratrol alleviates p-S396-tau hyperphosphorylation

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and oxidative damage in rat hippocampal slices exposed to vanadate, suggesting its potential

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application in sporadic AD therapeutics.

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Materials and methods

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Materials.

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Antibody against phosphoserine 396-Tau (p-S396-tau) was purchased from Invitrogen

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(invitrogen, Thermofisher scientific, Waltham, MA, USA). Antibodies against total and

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phosphorylated ERK (Thr201/Tyr204), p-AKT (Ser473), and Akt were obtained from Cell

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Signaling Technology (Beverly, MA, USA). Antibody against GSK-3 β Ser9 was purchased

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from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against GSK-3β Tyr216,

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GSK-3β, and Tau5 were from BD Bioscience (Franklin Lakes, NJ, USA). Secondary

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peroxidase-conjugated rabbit polyclonal and mouse monoclonal antibodies were obtained

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from Jackson Immuno Research (PA, USA). Secondary horseradish-peroxidase-conjugated

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goat polyclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA,

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USA). ERK inhibitor SL327, MAP kinase phosphatase 1 (MKP-1) inhibitor RO-318220,

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protein tyrosine phosphatase 1B (PTP1B) inhibitor, and Na3VO4, a broad-spectrum

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phosphatase inhibitor, were purchased from Merck Millipore (Merck KGaA, Darmstadt,

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Germany). ROS assay kit (STA-342) and LY294002 were obtained from Cellbiolabs (San

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Diego, CA, USA) and Abcam (Cambridge, MA, USA), respectively. LDH assay kit (TOX7),

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anti-β-actin antibody, resveratrol (trans-3,4′,5-trihydroxystilbene), glutathione (GSH), LiCl,

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FeCl2, and other chemicals were purchased from Sigma (St. Louis, Mo., USA).

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Hippocampal slice cultures.

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All experimental procedures were conducted via protocols approved by the Animal Care

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Committee of Ewha Medical Research Institute (EMS15-0323). In brief, hippocampal slice

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cultures were prepared from 10-day-old Sprague–Dawley rat pups (Harlan, Indianapolis, IN)

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as previously described.19,20 In hippocampal slices, the maturation of cells, synapses, and 6


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connectivity occurs as in their in vivo counterparts. Thus, these preparations are well suited

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for studies of prolonged pharmacological treatment and recovery, which would be difficult to

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perform in an intact animal system.

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Experimental treatment.

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Treatments were initiated at 10 days post-dissection. All reagents were added to serum-

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free medium (no horse serum) equilibrated at 37°C in 5% CO2 prior to addition to the slices.

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In brief, slices were pre-treated with a variety of pharmacological agents including SL327 (5

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µM), LiCl (from 2 to 5 mM) or LY294002 (5 µM) as described in the text. Na3VO4 was then

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added to the cultures in serum-free medium at various concentrations (from 50 to 200 µM) as

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noted in the text for 8 h. To determine the effects of MKP-1 or PTP1B, slices were treated

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with either RO-318220 or PTP1B inhibitor (40 µM each) for 8 h. All concentrations were

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selected on the basis of the maximal effects of the drugs on their specified targets. Vehicles

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were treated identically, but did not contain Na3VO4 or pharmacological agents as described

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above. Vehicle alone or pharmacological agents exerted no detectable effects on cell viability.

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At the indicated times after treatment initiation, the slices were rinsed twice with 1x

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phosphate-buffered saline and harvested via removing Millicell membrane inserts after

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freezing the samples on ice. They were then processed for immunoblotting as described

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below. To investigate the neuroprotective mechanism of resveratrol, slices were pretreated

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with resveratrol (20 µM), GSH (5 mM), SL327 (5 µM), or LiCl (5 mM) for 1 h and then

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further incubated with Na3VO4 (100 µM) or FeCl2 (100 µM) for 68 h. Culture media were

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collected to detect LDH leakage into the medium as a biomarker of hippocampal damage

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caused by Na3VO4 as described below.

167 7



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Quantification of cell death by lactate dehydrogenase activity.

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Lactate dehydrogenase (LDH) enzymatic activity in culture medium was used to

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determine the extent of cellular damage in cultured slices subjected to different treatments as

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previously described.25,26 Activities were expressed as the relative percentages using

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respective values for Na3VO4-treated slice culture set as 100%. The viability of hippocampal

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slices after Na3VO4 treatment was monitored by measuring the activity of LDH released into

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the incubation media. LDH activity was spectrophotometrically measured at 490–690 nm

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with a microplate reader (Labsystems iEMS reader MF) using a toxicology assay kit (Sigma,

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St. Louis, Mo., USA) according to the manufacturer instructions.

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Resveratrol treatment.

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Resveratrol was prepared in 50% dimethylsulfoxide (DMSO) to 100 mM. It was made

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fresh each time and diluted in culture medium to desired concentrations. Controls received

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the same amount of DMSO (final DMSO less than 0.1%). Hippocampal slices were

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pretreated with resveratrol (20 µM) for 1 h followed by further stimulation with Na3VO4 in

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serum-free medium at various time periods indicated. Slices were pretreated with resveratrol

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(20 µM/L) for 1 h, then SOV (100 µM) was added and incubated for another 8 h or 68 h.

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Detection of Reactive Oxygen Species (ROS).

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Fluorescent probes of 2′,7′-dichlorofluorescein diacetate (DCFH-DA) were employed in

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the determination of intracellular reactive species formation in hippocampal cells. To assess

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ROS-mediated oxidation of DCFH-DA to fluorescent compound, dichlorodihydrofluorescein

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(DCF), DCFH-DA in serum-free media was added to slices at a final concentration of 10 µM

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at 37°C for 30 min. Slices of each sample were then removed and homogenized in lysis 8


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buffer (ROS assay kit, STA-342). ROS production in the slice homogenates was quantified

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by measuring the formation of the fluorescence product of DCFH oxidation according to the

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manufacturer instructions.

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wavelength of 480 nm and an emission wavelength of 530 nm using a Synergy H1 (Biotek,

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Winooski, USA).

Fluorescence of oxidized DCF was measured at an excitation

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Western blotting.

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Immunoblotting was conducted as described previously.25,26 Briefly, equal quantities of

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sample proteins were separated by molecular weight on 10% SDS polyacrylamide gels and

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transferred onto PVDF membranes (GE Hearthcare, Sweden). After blocking with 3% milk in

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TBS-T for 30 min, the membranes were then probed with primary antibody diluted with 1%

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milk and incubated at 4 °C overnight. Signals were acquired with an ECL (GE Hearthcare)

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system after incubation with horseradish peroxidase-conjugated secondary antibodies.

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Quantification was performed by densitometric scanning of the film using a Las3000 Imaging

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Densitometer with Multi gauge 3.0 software (Fujifilm, Tokyo, Japan). The gray values of

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sample bands were quantified using U-SCAN-IT gel 6.1 and normalized against the levels of

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β-actin.

209 210

Statistical Analysis.

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Data are expressed as mean ± SEM of several experiments indicated. Differences between

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groups were examined for statistical significance using one-way analysis of variance

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(ANOVA) followed by Student’s t-test. Statistical significance was accepted at p < 0.05 level.

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Results 9



216 217

Hyperphosphorylation of tau at Ser396 site is significantly attenuated by ERK1/2

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inhibition in hippocampal slices exposed to Na3VO4.

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The increased level of phosphorylated tau at Ser396 (p-S396-tau) is a reliable marker of

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the severity of AD.12 It might be associated with protein phosphatase inhibition. To

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investigate whether Na3VO4 might induce hyperphosphorylation of tau and cause oxidative

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stress associated with AD-like damage in rat hippocampus, the levels of p-S396-tau protein

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were detected by Western blot using a tau antibody specific for the detection of p-S396-tau.

224

Hippocampal slices were treated with various concentrations (50-200 µM/L) of Na3VO4 for 8

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h and we found that Na3VO4 significantly increased the levels of p-S396-tau in a dose-

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dependent manner. However, total tau protein levels were not significantly altered (Figure 1A

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and Figure 1D). Based on the results, we selected 100 µM of Na3VO4 for 8 h treatment for

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the following experiments.

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MAPK/ERK, PI3K/AKT, and GSK-3β are three major protein kinases associated with

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tau pathology in AD. They could phosphorylate tau both in vitro and in vivo.21 To elucidate

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the mechanism of how Na3VO4 altered p-S396-tau levels, slices were exposed to Na3VO4 for

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8 h and assayed for Na3VO4-induced changes in the phosphorylation of the above three

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mentioned kinases. As shown in Figure 1, both phosphorylation levels of ERK1/2 and AKT

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were significantly elevated, while total ERK1/2 and AKT levels were not changed by

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Na3VO4 treatment (Figure 1B, 1C, 1E, and Figure 1F), indicating that increased kinase

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activities might have contributed to tau hyperphosphorylation following Na3VO4 insult.

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However, GSK-3β phosphorylation at Tyr216 site (functional activation) and protein

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expression were not significantly altered after Na3VO4 treatment (Data not shown).

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We further analyzed whether inhibition of activities of either ERK1/2 or AKT would 10


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reduce the level of p-S396-tau following Na3VO4 insult. Functional inhibition of ERK1/2

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using a specific inhibitor SL327 capable of crossing the BBB potently attenuated Na3VO4-

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mediated increase of p-S396-tau levels (Figure 2A, 2C, 2D, and Figure 2F). In contrast,

243

LY294002 that significantly repressed the increase in PI3K/AKT activation did not suppress

244

Na3VO4-elicited changes in p-S396-tau levels (Figure 2B, 2C, 2E, and Figure 2F), indicating

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that Na3VO4-induced tau S396 phosphorylation was independent of PI3K/AKT activation.

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These results suggest that activation of ERK1/2 is at least in part responsible for the Na3VO4-

247

elicited increases in p-S396-tau levels and that down-regulation of p-ERK1/2 reduces p-

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S396-tau levels in Na3VO4-treated hippocampal slices.

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Inactivation of MKP-1 and PTP1B elicits hyperphosphorylation of tau at S396 in

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hippocampal slices.

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MAPK phosphorylation is a reversible process controlled tightly by the activity of protein

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phosphatases. Archetypal deactivator of MAPKs is MAP kinase phosphatase 1 (MKP1), also

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known as known as DUSP1. MKP1 is a member of a family of dual-specificity phosphatases

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that dephosphorylate both threonine and tyrosine residues. Therefore, MKP-1 serves as a key

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negative regulator of MAPK cascade.28 To elucidate the potential targets of Na3VO4 in rat

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hippocampal slices, effects of RO-318220 (MKP-1 inhibitor) on both ERK1/2

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phosphorylation status and p-S396-tau levels were evaluated. As shown in Figure 3, RO-

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318220 replicated Na3VO4-induced changes, increasing ERK1/2 activation and p-S396-tau

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levels in hippocampal slices. Similarly, a specific inhibitor of protein tyrosine phosphatase 1B

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(PTP1B) had comparable effects. These results confirmed that repression of phosphatases

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such as MKP-1 and PTP1B was associated with ERK1/2 activation, consequently increasing

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p-S396-tau levels in hippocampal slices exposed to Na3VO4. 11



264 265

Resveratrol attenuates Na3VO4-induced hyperphosphorylation of tau at S396 via

266

decreasing ERK1/2 activation.

267

Despite numerous studies have revealed beneficial effects of resveratrol on Aβ pathology

268

of AD, the effect of resveratrol on Na3VO4-induced tau phosphorylation at Ser396 and the

269

underlying signaling pathways remain unclear. Given that suppressing ERK1/2 activity could

270

attenuate tau phosphorylation in Na3VO4-treated slices, we next determined whether

271

resveratrol could decrease the activity of ERK1/2 and attenuate Na3VO4-induced P-S396-tau

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hyperphosphorylation under the same experimental paradigm. We observed that resveratrol

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significantly inhibited the increase in ERK1/2 activation by Na3VO4 treatment (Figure 4A

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and Figure 4D). Furthermore, application of resveratrol resulted in a significant decrease in

275

the levels of p-S396-tau induced by Na3VO4 insult (Figure 4B and Figure 4E). This result

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indicates that resveratrol can effectively suppress Na3VO4-induced changes of ERK1/2

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activation and increase of p-S396-tau levels, further confirming that ERK1/2 activation is

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related to tau hyperphosphorylation at Ser396 in Na3VO4-treated hippocampal slices

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Resveratrol increases the inhibitory GSK-3β β Ser9 phosphorylation required for

281

lowering p-S396-tau levels in Na3VO4-treated slices.

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GSK-3β activity is regulated by Tyr216 (stimulatory) and Ser9 (inhibitory)

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phosphorylation. Decreased GSK-3β Ser9 phosphorylation has been reported to be associated

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with AD-like tau phosphorylation.27 Therefore, we determined whether resveratrol could

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modulate GSK-3β activity by immunoblot analysis using specific activity-dependent

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phospho-antibodies. Indeed, resveratrol significantly increased GSK-3β Ser9 phosphorylation

287

in Na3VO4-treated slices (Figure 4C and Figure 4F), indicating that resveratrol could reduce 12


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GSK-3β activity. Having found that resveratrol could reduce GSK-3β activity, we next set out

289

to evaluate if this increase of GSK-3β Ser9 phosphorylation was associated with reduced

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levels of p-S396-tau in Na3VO4-treated slices. Pharmacological inhibition of GSK-3β with

291

LiCl obviously increased GSK-3β Ser9 phosphorylation (Figure 5A and Figure 5D), resulting

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in reduced p-S396-tau levels (Figure 5B and Figure 5E). These results are similar to the

293

results obtained with resveratrol, indicating that the reduction of GSK-3β activity through

294

increased GSK-3β Ser9 phosphorylation is also required for lowering p-S396-tau levels in

295

Na3VO4-treated slices. In addition, SL327 attenuated Na3VO4-induced increase of p-S396-tau

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through downregulating p-ERK1/2 cascade (Figure 2) and elicited significant increase of

297

GSK-3β Ser9 phosphorylation (Figure 5C and Figure 5F), whereas LiCl concentrations up to

298

5 mM failed to significantly affect the phosphorylation status of ERK1/2 (data not shown).

299

These results confirmed that resveratrol’s downregulation of ERK1/2 activation was followed

300

by an increase of GSK-3β Ser9 phosphorylation, which ultimately alleviated p-S396-tau

301

levels in Na3VO4-treated hippocampal slices.

302 303

Resveratrol can decrease the hippocampal toxicity resulting from ROS generation after

304

long term exposure to Na3VO4.

305

Besides protein phosphatase inhibition, Na3VO4, a pentavalent vanadium compound,

306

could induce oxidative damage to critical biomolecules and consequently induce

307

hippocampal injury like other free redox active metals such as Fe2+.29,30 Recent report also

308

implicated that vanadium compounds might be subject to Fenton reaction causing oxidative

309

stress.24 Therefore, we evaluated the protective effect of resveratrol on hippocampal toxicity

310

due to oxidative stress after a long-term exposure to Na3VO4. Notably, resveratrol

311

pretreatment for 1 h before Na3VO4 insult caused an attenuation of Na3VO4-mediated 13



312

hippocampal toxicity resulted from exposure of slices to 100 µM Na3VO4 for 68 h based on

313

LDH release assay (Figure 6A). We further determined the involvement of reactive oxygen

314

species (ROS) in Na3VO4-induced hippocampal neurotoxicity. As shown in Figure 6B,

315

Na3VO4 treatment significantly increased the extracellular levels of ROS compared to the

316

control group. Resveratrol markedly reduced such increase. Besides resveratrol, glutathione

317

(GSH), an important antioxidant in the brain, was also highly effective in reducing

318

extracellular ROS level upon Na3VO4 insult (Figure 6B). It could obviously protect

319

hippocampal slices against Na3VO4-mediated toxicity (Figure 6A). In contrast, both SL327

320

and LiCl failed to decrease this Na3VO4-induced ROS generation and they elicited no

321

protection against Na3VO4 (data not shown). Moreover, resveratrol and GSH potently

322

reduced Fe2+ metal ion-induced hippocampal toxicity by relieving the upregulation of

323

extracellular ROS generation (Figure 6C and Figure 6D). In contrast to resveratrol, GSH

324

failed to significantly lower the level of p-S396-tau in hippocampus exposed to Na3VO4 (data

325

not shown). Thus, resveratrol and GSH diminished the increased extracellular ROS

326

generation and hippocampal toxicity upon a long-term exposure to Na3VO4 or FeCl2. Such

327

attenuation at least in part was dependent on its anti-oxidative activities, independent of the

328

regulation of ERK1/2 or GSK-3β signaling cascades involved in tau hyperphosphorylation in

329

hippocampus.

330 331

Discussion

332

This study generated five primary findings regarding the underlying molecular

333

mechanisms of resveratrol against tau hyperphosphorylation and oxidative damage, two

334

major pathological features of AD, following vanadate insult in rat hippocampal slices. First

335

resveratrol significantly inhibited Na3VO4-induced increase of p-S396-tau levels selectively 14


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upregulated in the hippocampus of AD brains. Second, this inhibitory effect of resveratrol

337

was mediated by the downregulation of Na3VO4-induced ERK1/2 activation mimicked by

338

SL327. Third, repression of phosphatases such as MKP-1 and PTP1B replicated this Na3VO4-

339

induced changes, increasing both ERK1/2 activation and the p-S396-tau levels. Fourth,

340

resveratrol also potently induced an inhibitory GSK-3β Ser9 phosphorylation, strengthening

341

the inhibitory effect of resveratrol on p-S396-tau hyperphosphorylation, which was replicated

342

by the pharmacologic inhibition of GSK-3β with LiCl. Finally, resveratrol diminished the

343

increased extracellular ROS generation and hippocampal toxicity upon a long-term exposure

344

to Na3VO4 or FeCl2. These findings strongly suggest that resveratrol could be used as a major

345

compound in AD treatment.

346

Tau phosphorylation at Ser396 site has been postulated to be able to reduce tau-

347

microtubule binding as an early key step in the development of tau pathology in AD brains.13

348

Recent studies have reported that hyperphosphorylated tau on Ser396 detected in the

349

hippocampus is associated with cognitive decline in AD model rats.31,32 In fact, tau

350

hyperphosphorylation at Ser396 in the brain of 5XFAD mice is significantly higher than that

351

in wild-type mice in the hippocampus. This occurs at a much earlier stage than the

352

appearance of learning and memory disorders.33 Recent study has also shown that tau

353

phosphorylation of tau at Ser396 site can lead to impaired associative memory which relates

354

the best to disease progression.34 Tau phosphorylation at S396 (not S404) within the PHF-1

355

epitope is critical for the induction of long-term depression of synaptic transmission in the

356

hippocampus.35 These findings implicate that accumulation of hyperphosphorylated tau at

357

S396 over time may induce the decrease in cognition and learning disabilities possibly

358

through a deficit in spatial reversal learning memory. Therefore, a therapeutic molecule that

359

can reduce this pathogenic tau phosphorylation at Ser396 might effectively interfere with the 15



360

progression of AD.

361

Of note, the present study clearly demonstrated that vanadate significantly induced a

362

pathogenic hyperphosphorylation of tau at S396 site. Such hyperphosphorylation of tau was

363

preferentially attenuated by resveratrol in the hippocampus, a critical step in preventing and

364

slowing down AD pathology. Furthermore, for the first time, we showed that the

365

aforementioned effect of resveratrol was at least in part mediated by downregulating ERK1/2

366

activation through repressing phosphatases such as MKP-1 and PTP1B in Na3VO4-treated

367

hippocampal slices. This is in good agreement, at least in part, with our previous study

368

demonstrating that resveratrol can down-regulate ERK1/2 activation, thereby decreasing

369

monocyte chemoattractant protein-1 expression and the resulting IL-1β levels, depicting the

370

anti-neuroinflammatory properties of resveratrol via ERK1/2 inactivation.26 Consistently,

371

earlier report demonstrating that resveratrol attenuated ERK signaling and inhibited incision-

372

induced pain in a model of incision-induced allodynia in mice36 adds strong support to our

373

findings. Notably, increase of MKP-1 phosphatase has been recently postulated as an anti-

374

inflammatory target for the treatment of chronic neuronal diseases.37 Moreover, PTP1B has

375

been implicated in activation of protein phosphatase-2A (PP2A),38 which is significantly

376

reduced in AD brains.39 Therefore, our findings that repression of MKP-1 and PTP1B

377

resulted in an increase of p-S396-tau levels through ERK1/2 activation in the hippocampus

378

suggested that upregulation of MKP-1 and PTP1B by pharmacological means might

379

represent a potential strategy in arresting tau pathology in AD.

380

GSK-3β serves as a key target for identifying drugs for treating AD by alleviating tau

381

aggregation. Therefore, compounds producing a therapeutic effect by attenuating tau

382

hyperphosphorylation at multiple AD-related sites through upregulating inhibitory Ser9-

383

posphorylated GSK-3β are actively being sought.40,41 In this regard, it is interesting to note 16


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384

that resveratrol and SL327 could reduce Na3VO4-induced ERK1/2 activity, increase

385

inhibitory GSK-3β Ser9 phosphorylation, and consequently decrease Na3VO4-induced p-

386

S396-tau levels as conformed by pharmacologic inhibition of GSK-3β with LiCl.

387

Accordingly, the present study clearly demonstrated that resveratrol could downregulate p-

388

S396-tau levels in vanadate-treated hippocampus through inhibiting the activation of ERK1/2

389

and GSK-3β, the two main tau kinases. These results strongly suggest that resveratrol could

390

be used as a potential candidate for rescuing p-S396-tau pathology and ensure delayed

391

disease progression of MCI to AD dementia.14

392

Importantly, a recent study demonstrating that drugs inhibiting GSK-3β might prove

393

beneficial for controlling cognitive impairments caused by excessive stress and the associated

394

neuroinflammation42 strongly support our study implicating that resveratrol could serve as a

395

potential nutraceutical agent for arresting tau pathology in AD. On the other hand, one study

396

has shown that inhibition of PI3K/AKT and increased GSK-3β activation can lead to

397

hyperphosphorylation of tau protein and the accumulation of NFTs.43 However, no clear

398

modification of tau phosphorylation at Ser396 by increased PI3K/AKT activity following

399

vanadate insult was observed in this study. Earlier study also reported that resveratrol

400

activated both Akt and ERK signaling pathways, which functioned to inactivate GSK-3β and

401

to promote angiogenesis in human endothelial cells.44 Some of these discrepancies may

402

depend on the stimulants and the concentrations used and the different tissues or cells.

403

Interestingly, one report has recently described that resveratrol decreased ERK and GSK-3β

404

phosphorylation in early passage mesenchymal stem cells expressing SIRT1 whereas it

405

increased ERK and GSK-3β phosphorylation in late passage cells, which did not express

406

SIRT1.45

407

To the best of our knowledge, the present study is the first one demonstrating that a 17



408

long-term exposure to Na3VO4 can promote extracellular ROS generation, consequently

409

leading to hippocampal toxicity. Consistently, earlier study clearly demonstrated that the

410

genotoxicity of pentavalent vanadium such as Na3VO4 was expressed in vivo only following

411

high dose exposure possibly as a consequence of poor bioavailability of the element.46 Recent

412

study also demonstrated that lethargy and abnormal gait were observed in high dose

413

vanadate-exposed animals.47 The contribution of brain metal dyshomeostasis including free

414

redox active metals such as Fe2+ to the onset and/or progression of AD has been well

415

documented in several in vitro and in vivo studies.30 Thus, further study is needed to establish

416

the possible role of vanadium compound in the pathogenesis of sporadic AD with a cautious

417

approach.

418

Several reviews have highlighted the potential neuroprotective effects of resveratrol in

419

AD via different mechanisms, most notably the activation of NAD(+)-dependent histone

420

deacetylases enzymes termed sirtuins including SIRT1, and AMPK pro-survival

421

pathways.15,16 Of note, resveratrol exerts neuroprotective effects in rats from Aβ-induced

422

neurotoxicity by reducing the inducible nitric oxide synthase and lipid oxides, thereby

423

reducing memory loss.48 Consistently, the present study showed that resveratrol protected

424

against hippocampal toxicity by relieving the upregulation of extracellular ROS generation

425

upon treatment with the pentavalent species of vanadium (vanadate) such as Na3VO4 or iron

426

(Fe2+) metal compounds. GSH, a major brain antioxidant that prevents damage to cellular

427

components caused by ROS such as free radicals and peroxides, markedly mimicked

428

resveratrol’s protection against the hippocampal toxicity mediated by these metal compounds,

429

relieving the upregulation of extracellular ROS generation. However, no clear protection

430

against extracellular ROS generation and hippocampal toxicity following a long term

431

exposure to Na3VO4 was observed by SL327 or LiCl. Therefore, long-term resveratrol 18


Page 18 of 34

Page 19 of 34


432

treatment-driven reductions on hippocampal toxicity in the parameters studied here could be

433

mediated by the well-known antioxidant effects of this polyphenol rather than through its

434

inhibitory effects on tau kinases.

435

Apart from the antioxidative potential, resveratrol also maintains the integrity of BBB.49

436

It can enhance the formation of long-term memory and the strength of LTP by increasing

437

brain-derived neurotrophic factor which repairs cognitive loss and ameliorates damaged

438

hippocampal circuits, thus restoring memory and cognition.50 Resveratrol also protects

439

neurons against Aβ-induced disruption of spatial learning, memory and hippocampal LTP

440

through the mechanisms involving rescue of SIRT1 expression and CREB phosphorylation in

441

rats.51 Consistently, earlier studies reported that resveratrol reduced oxidative stress by

442

activating SIRT1 in hippocampus52 and neuronal cells.53 In contrast, recent study has shown a

443

SITR1-independent attenuation of oxidative stress by resveratrol in skeletal muscle cells.54

444

Thus, these findings together strongly support that the multiple potential mechanisms of

445

action of resveratrol at a multitude of molecular targets associated with the pathobiology of

446

AD would be beneficial to exploit it as a multi-target directed drug for battling AD.8,9,55 More

447

work is required to elucidate the mechanisms underlying an intricate signaling crosstalk by

448

resveratrol between SIRT1 activation and ROS reduction as well as inactivation of

449

ERK/GSK-3β signaling cascades in the hippocampus exposed to vanadate.

450

In spite of the considerable advances in clarifying the multiple mechanisms of actions of

451

this multitarget resveratrol, a phase II randomized, double-blind, placebo-controlled trial of

452

resveratrol for AD has recently shown unnecessary side effects from daily intake of

453

resveratrol including nausea, diarrhea, and weight loss.18 Therefore, further studies with

454

resveratrol derivatives and analogs that show better responses with less toxicity than

455

resveratrol itself56 or resveratrol combination therapy as being recently shown57,58 might 19



456

provide renewed insight on the exact clinical effects of resveratrol in treating multifactorial,

457

complex etiological AD.

458

In conclusion, present study indicated that vanadate significantly induced tau

459

hyperphosphorylation and oxidative stress, which are associated with the major pathological

460

changes of AD, in rat hippocampal slices. Furthermore, resveratrol alone not only decreased

461

tau hyperphosphorylation at Ser396 site, a potential target for delaying progression of MCI to

462

AD dementia, via downregulating ERK1/2 and GSK-3β signaling cascades, but also rescued

463

the hippocampus from vanadate-induced oxidative damage via reducing extracellular ROS

464

levels. Therefore, resveratrol might have preventive and/or therapeutic potential for AD

465

management, although the relevance of these in vivo findings remains to be clearly elucidated.

466 467

Abbreviations: AD: Alzheimer's disease; Aβ: amyloid-β; ΒΒΒ: blood-brain barrier;

468

H2DCFDA: 2′7′-dichlorodihydrofluorescein diacetate; DMSO: dimethylsulfoxide; ERK1/2:

469

extracellular signal-regulated kinase1/2; GSH: glutathione; GSK-3β: glycogen synthase

470

kinase-3β; LDH: Lactate dehydrogenase; MCI: mild cognitive impairment; MKP1: MAP

471

kinase phosphatase 1; NFTs: neurofibrillary tangles; PI3K: phosphoinositide 3-kinase; p-

472

S396-tau: phosphorylated tau protein at Ser396 site; PTP1B: protein tyrosine phosphatase 1B;

473

ROS: reactive oxygen species

474 475

Funding sources

476

This research was supported by the Basic Science Research Program through the National

477

Research Foundation (NRF) and funded by the Ministry of Science, ICT & Future Planning,

478

Republic of Korea (Grant No. NRF-2010-0027945 & NRF-2015R1A2A2A0100 5226).

20


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Figure legends

654 655

Figure 1. Effects of Sodium orthovanadate (Na3VO4) on tau hyperphosphorylation at Ser396

656

site and the activation of ERK1/2, PI3K/AKT, and GSK-3β signaling pathways in rat

657

hippocampal slices. After slices were treated with Na3VO4 for 8 h at the indicated

658

concentrations, the extracts of slices were prepared. (A) Protein levels of tau phosphorylation

659

at Ser396 site (p-S396-Tau) were detected by Western blot analysis with a site-specific

660

primary antibody. The levels of total tau protein were monitored with Tau-5 antibodies. The

661

protein levels of phosphorylated ERK1/2 and total ERK1/2 (B), and phosphorylated AKT and

662

total AKT (C) were also measured by Western blot analysis. Levels of β-actin were examined

663

to ensure that equal amounts of proteins were loaded. (D, E, F) Densitometric analysis of A,

664

B, C. All data are presented as means ± standard errors (n = 3–5). *P

Resveratrol Ameliorates Tau Hyperphosphorylation at Ser396 Site

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