Dietary Lycopene Supplementation Improves Cognitive Performances in Tau Transgenic Mice Expressing P301L Mutation via Inhibiting Oxidative Stress and Tau Hyperphosphorylation

Abstract

Background: Oxidative stress is implicated in the pathogenesis of Alzheimer’s disease (AD) and other tauopathies and participates in their development by promoting hyperphosphorylation of microtubule-associated protein tau. Lycopene, as an effective antioxidant, combined with vitamin E seemed to be additive against oxidative stress.

Objective: The present study was undertaken to examine whether lycopene or lycopene/vitamin E could exert protective effects on memory deficit and oxidative stress in tau transgenic mice expressing P301L mutation.

Materials and methods: P301L transgenic mice were assigned to three groups: P301L group (P301L), P301L+lycopene (Lyc), and P301L+lycopene/vitamin E (Lyc+VE). Age-matched C57BL/6J mice as wild type controls (Con) were used in the present study. Spatial memory was assessed by radial arm while passive memories were evaluated by step-down and step-through tests. Levels of tau phosphorylation were detected by western blot. Oxidative stress biomarkers were measured in the serum using biochemical assay kits.

Results: Compared with the control group, P301L mice displayed significant spatial and passive memory impairments, elevated malondialdehyde (MDA) levels and decreased glutathione peroxidase (GSH-Px) activities in serum, and increased tau phosphorylation at Thr231/Ser235, Ser262, and Ser396 in brain. Supplementations of lycopene or lycopene/vitamin E could significantly ameliorate the memory deficits, observably decreased MDA concentrations and increased GSH-Px activities, and markedly attenuated tau hyperphosphorylation at multiple AD-related sites.

Conclusions: Our findings indicated that the combination of lycopene and vitamin E antioxidants acted in a synergistic fashion to bring significant effects against oxidative stress in tauopathies.

Keywords

Lycopene oxidative stress P301L tau protein phosphorylation spatial memory vitamin E

INTRODUCTION

Hyperphosphorylation and aggregation of the microtubule-associated protein tau in neurons are pathological hallmarks of a large family of neurodegenerative disorders, named tauopathies including Alzheimer’s disease (AD) [1]. AD is the most common neurodegenerative disorder affecting elderly people, gradually declining in cognitive function and memory loss [2]. Hyperphosphorylated tau is prone to misfold and aggregate the intracellular neurofibrillary tangles (NFTs), eventually leading to the disruption of neuronal transport and the degeneration of the affected neurons [3]. In addition, oxidative stress is another characteristic of tauopathies in addition to NFTs [4]. Substantial evidence supports that the presence of extensive oxidative stress is associated with tauopathies of AD patients [5], frontotemporal lobar degeneration disorders [6], and progressive supranuclear palsy types [7].

Lycopene is a red plant pigment found in tomatoes, apricots, watermelons, etc. As one of the carotenoids, it is an effective antioxidant with a singlet-oxygen-quenching capacity 47 and 100 times stronger than that of vitamin E and beta-carotene, respectively [8]. Clinical study indicated that high serum levels of lycopene were associated with a lower risk of AD mortality in older adults [9]. It has been shown that AD patients have low levels of circulating lycopene compared to healthy controls [10, 11]. Vitamin E is another important antioxidant primarily protecting cells from oxidative damage caused by free radicals. In comparison with groups treated with lycopene or vitamin E alone, the intervention studies using combinations of antioxidants seem to be additive in the co-treated group [12–14]. Although the data from cross-sectional and longitudinal studies on antioxidants and cognitive function are still conflicting [15], there are few findings in relation to lycopene and cognitive function. Moreover, lycopene shows positive effects as an antioxidative treatment in prostate cancer [12]. Based on the fact that oxidative stress is involved in tauopathy-associated neurodegenerative disease, lycopene may eventually bear fruit in the form of an effective approach to treat AD and/or related disorders [16].

With this background, the present study was undertaken to investigate the neuroprotective effects of lycopene on oxidative stress, tau phosphorylation, and memory in tau transgenic mice expressing the P301L mutation. Additionally, the synergistic effect of lycopene in combination with vitamin E has also been determined in our study.

MATERIALS AND METHODS

Antibodies and chemicals

Anti-tau monoclonal antibody (mAb) tau46 and tau13, respectively, detecting total tau and human tau, were purchased from Santa Cruz Biotechnology. Anti-β-tubulin polyclonal antibody was purchased from Abcam Ltd. The following phosphorylation-dependent tau antibodies were used: AT180 to detect tau phosphorylated at Thr231 and Ser235 (Thermo Scientific); pS396, pS262 for tau phosphorylated at Ser396, Ser262 respectively (all from Abcam Ltd). As secondary antibodies, HRP-conjugated goat anti-rabbit IgG or goat anti-mouse IgG were used (Sigma Aldrich). Enhanced ChemoLuminescence system (ECL) was purchased from Advansta Inc. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) kits were bought from Nanjing Jiancheng Bioengineering Institute.

Lycopene and vitamin E source

The lycopene oleoresin and natural vitamin E acetate were provided by BY-HEALTH Co., Ltd. Lycopene preparations and compound preparations (containing lycopene and vitamin E) were mixed with salad oil and stored at 4°C in the dark.

Animals and experimental design

Male P301L transgenic mice (25∼28g in weight and 7 months±10 days old) and age-matched C57BL/6J mice as wild type controls were used in the present study. P301L transgenic mice were kindly offered by Prof. Xuelan Wang (Zhongshan School of Medicine, Sun-Yet Sen University) [17]. Transgenic tau expression is under the control of a modified version of the neuron-specific mThy1.2 promoter [18]. All mice were kept in individually ventilated cages (3 mice per cage) with ad libitum access to irradiated chow and acidified drinking water under a controlled environment (22–25°C and 50±5% relative humidity, 12-h light-dark cycle). All animal experiments were performed in accordance with the Policies on the Use of Animals and Humans in Neuroscience Research (1995), and the experimental protocols were approved by Institutional Animal Ethics Committee.

Forty-six P301L mice were randomly divided into 3 groups (P301L, n = 15; Lyc, n = 15; Lyc+VE, n = 16) and respectively treated with salad oil, 5 mg*(kg.bw)^-1 lycopene, and 5 mg*(kg.bw)^-1 lycopene in combination with 3 mg*(kg.bw)^-1 vitamin E by gavage for 8 weeks. C57BL/6J mice (n = 12) as wild type controls were supplemented with salad oil. From the ninth week, behavior performances were determined. Daily food consumption and body weight were recorded oncea week.

Radial arm maze test

The procedures of the eight-arm maze performance were previously described [19]. Briefly, mice were food-restricted to 80% ∼85% of their normal body weight, which was maintained throughout the test. During the pre-training session, the food reward (peanut) was respectively placed in holes at the end of each arm to ensure that mice learned to explore the food bait and habituated to the maze for 8 min.

8-arm maze testing occurred for 9 consecutive days, one trial per day. During testing, animals were placed onto the center platform and were allowed 8 min to retrieve all peanuts in four (arms 1, 3, 5, and 6) of the eight arms. After eating all 4 cereal baits or once 8 min had elapsed, animals were placed in their home cage. Memory acquisition was assessed as the decrease in the number of errors and retrieving time between each trial. Reference memory errors were scored as entries into a non-baited arm. Working memory errors were scored as reentries into an arm from which food had already beeneaten.

Step-through passive avoidance test

The step-through passive avoidance task was conducted as previously reported [19]. On the first day, mice were allowed to move freely in a step-through apparatus (equally divided into one light and one dark compartment) for 5 min. On the second day, the mice were briefly placed in the light compartment back to the door leading to the adjoining darkened compartment. Once inside the dark compartment after the door was opened, the mice received a brief electrical stimulation (2 mA, 3 s) delivered from the metal floor. On the third day, when placed inside the apparatus again, mice that recalled the aversive experience in the dark compartment will avoid entering the dark during testing. The latencies and avoidance of the dark compartment wererecorded.

Step-down passive avoidance test

The step-down passive avoidance task utilizes a mouse’s desire to step-down off of a small circular rubber platform (Diameter at the bottom: 4 cm; Diameter at the top: 3.4 cm; Height: 3 cm) onto the comfortable metal floor of the testing apparatus (STT-2, Institute of Materia Medica Chinese Academy of Medical Sciences). However, the shock from the metal floor requires that the animal inhibit its instinctive behavior. On the first day of training, the mice were shocked (2mA, 3s) only after they stepped down off a platform. On the next day, mice that had a good memory stayed on the small platform for a longer time before stepping down. The latency to step down was used to assess passivememory.

Biochemical assay

Blood samples were collected from the orbit of mice before dislocation and promptly centrifuged at 3,000 g for 10 min, and the supernatant (serum) was store at –80°C for the determination of indicators of oxidative stress (GSH-Px, SOD, and MDA). SOD activity was determined by the inhibition of nitrobluetetrazolium (NBT) reduction due to O₂^- generated by the xanthine oxidase system. One unit of SOD activity was defined as the amount of protein causing 50% inhibition of the NBT reduction rate. The reaction product was evaluated spectrophotometrically at 550 nm. GSH-Px activity was assayed using glutathione (GSH) as substrate. The 1μmol/LGSH disappearance at 412 nm was one unit of enzymatic activity. The level of lipid peroxidation was assessed by measuring thiobarbituric acid reactive substances (TBARS) and the product was evaluated spectrophotometrically at 532 nm.

Western blotting

Mice were dislocated after anesthesia, and the brain tissues were rapidly removed and stored at –80°C. Western blotting was performed according to methods established in our laboratory [19]. The protein bands were visualized with enhanced chemiluminescence and quantitatively analyzed by Gel-pro analyzer software (version 4.0, Media Cybernetics, L.P.).

Statistical analysis

Results are presented as mean±SEM or mean±SD. All data were analyzed by t-test, Mann-Whitney test or repeated measures analysis of variance (ANOVA) with SPSS (version 21.0, Chicago, USA), followed by the least significant difference (LSD) post hoc test to compare differences between groups. In all cases, p < 0.05 was considered statistically significant.

RESULTS

Lycopene ameliorates memory deficits of P301L mice

During the 9-day learning process in the 8-arm maze, P301L mice spend more time to find peanuts (F(1,7) = 17.85, p = 0.004; Fig. 1A) and made more reference memory errors (F(1,7) = 6.58, p = 0.037; Fig. 1B) and working memory errors (F(1,7) = 9.29, p = 0.019; Fig. 1C), which meant that spatial memory of the P301L mice had been impaired. Mice treated with lycopene showed less exploring time (p < 0.05, Fig. 1A), and fewer memory errors (p < 0.05, Fig. 1B, C) than P301L mice. Similarly, lycopene and vitamin E combinations shortened seeking time (p < 0.01, Fig. 1A) and decreased reference and working memory errors (p < 0.01, Fig. 1B,C). Compared with Lyc groups, Lyc+VE mice spent less time exploring to find the bait (p < 0.05, Fig. 1A).

We also performed step-down and step-through tests on mice to assess their passive learning and memory. After 24 h training in the step-down test, P301L mice displayed a significantly shorter latency to step off the rubber platform compared to the control mice (control mice:182.5±46.6 s, P301L mice:15.3±7.5 s, p < 0.01; Fig. 2A). Likewise, for the step-through passive performance, P301L entered the dark compartment more rapidly than control mice (control mice: 185.2±33.2 s, P301L mice: 62.7±15.0 s, p < 0.05; Fig. 2B). And yet, treatment with lycopene or lycopene/vitamin E significantly increased step-down latency of P301L mice (lyc group: 89.3±42.5 s, p < 0.05; lyc+VE group: 139.9±48.3 s, p < 0.01; Fig. 2A). Similarly, in step-through performance, lycopene or lycopene/vitamin E supplementations observably extended latency to enter the energized dark compartment (lyc group: 147.8±22.7 s, p < 0.05; lyc+VE group: 189.7±42.5 s, p < 0.05; Fig. 2B).

Lycopene attenuates oxidative stress in serum of P301L mice

There were no significant differences on SOD activities between groups (Table 1). Increase in serum MDA concentration (p < 0.05) and decrease in serum GSH-Px activity (p < 0.05) were exhibited in P301L mice in comparison to controls. However, increase in GSH-Px activity and decrease in MDA level in serum of P301L mice responded to dietary lycopene (p < 0.05) or lycopene/vitamin E (p < 0.01). Compared with the Lyc group, higher GSH-Px activity and lower MDA level were observed in mice of the lyc+VE group (p < 0.05).

Lycopene prevents tau hyperphosphorylation in brain of P301L mice

Figure 3 showed that human tau including mutation P301L was only detected by antibody tau13 in P301L transgenic mice but not in wild type mice (referred to “Con” group). To determine the effects of lycopene or lycopene/vitamin E on tau phosphorylation of P301L mice, we measured the phosphorylation at Thr231/Ser235 (AT180), Ser262, and Ser396 epitopes in brain by western blot analysis (Fig. 3A). The results showed that the expressions of total tau between the four groups were not significantly different (Fig. 3B). A significant elevation of the phosphorylated tau at Thr231/Ser235, Ser262, and Ser396 was detected in P301Lmice (Fig. 3C), which suggested that the human tau-transgenic mice expressing P301L mutation displayed AD-like tau hyperphosphorylation. However, simultaneous supplement of lycopene attenuated the tau hyperphosphorylation at Ser262 epitope but not Thr231/Ser235 and Ser396 epitopes. Furthermore, combined intake of lycopene and vitamin E decreased the levels of both Ser262 and Ser396 phosphorylation. Moreover, the phosphorylated level of Ser396 in mice of Lyc+VE group was significantly lower than Lyc group.

DISCUSSION

Transgenic mouse models of tauopathy have been used to further investigated the role of hyperphosphorylated tau and its effects on the neurodegeneration [20]. P301L transgenic mice, which carry the human tau gene with the P301L mutation, develop tau pathology in the brain [17]. In the present study, we used P301L mice to evaluate the antioxidant effects of lycopene or lycopene/vitamin E in relationship to memories and tau phosphorylation.

In P301L mice, elevated tau phosphorylation was observed in the brain compared to C57BL/6J mice. Similar results have been documented previously in P301L mice as a hallmarker of tauopathy [21]. Hyperphosphorylated tau is prone to misfold and aggregate to NFTs, which could be easier to prompt oxidative stress [22]. It is reported that cortical neurons expressing truncated tau show increased levels of reactive oxygen species [23]. Blood biomarkers of oxidative stress reflect such stress in the brain [24], and have been identified in AD patients or related animal models, including end-products of lipid peroxidation, such as malondialdehyde (MDA), ketones, epoxides and isoprostanes [25]. Increased levels of MDA in the brain of AD and mild cognitive impairment (MCI) have been confirmed by several studies [26]. Apart from the intracellular accumulation of peroxidation products, decreases in the activities of antioxidant enzymes, such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), have been observed in brain tissues of AD and MCI patients [27, 28].

Similar to the above-mentioned studies, we identified that P30L mice suffered from strong oxidative stress, which led to lower GSH-Px activity and higher lipid peroxide MDA in serum, in turn eventually leading to aggravate tau hyperphosphorylation [29]. GSH-Px represents an important constituent of the cellular defense mechanism against oxidative stress and is responsible for the metabolism of lipid peroxide [30]. In a nutshell, both decrease in the cellular defense (GSH-Px activity) and increase in oxidative stress (MDA level), contributing to the pathogenesis of tauopathies. In a Drosophila model expressing human tau with mutation R406W, tau-induced neurodegenerative abnormalities were proven to be mediated by oxidative stress [31]. Similarly, cellular experimental data suggested that chronic oxidative stress facilitates tau hyperphosphorylation [32]. Furthermore, the lipid peroxidation product, 4-hydroxy-2-nonenal, and acrolein could potentiate phosphorylated tau to form polymersin vitro and in vivo. Thus, the vicious cycle, where tau hyperphosphorylation and oxidative stress interact as both cause and effect, ultimately results in a decline of cognitive function, in line with our results that P301L mice had behavioral deficits in passive and spatial memories. Yet, the underlying mechanisms between oxidative stress and tau phosphorylation need to be further studied. The findings by Dumont et al. suggest that oxidative stress and mitochondrial abnormalities appeared prior to tau pathology, leading to behavioral dysfunction and neurodegeneration [35]. Indeed, mitochondria play a key role in the antioxidant response [36]. As evidenced by Dumont et al. in P301S mice, the dysregulations of key mitochondrial enzymes were indicated in tauopathy [35].

Lycopene is a strong antioxidant with an ability to reduce oxidative damage to lipids, proteins, and DNA [37]. Pure lycopene or tomato extracts was able to rescue cells from oxidative stress injury [38, 39]. In the present study, lycopene supplementation significantly elevated the activity of GSH-Px and decreased the MDA in serum, probably due to its antioxidant nature (Table 1). Furthermore, a more significant extent was found after lycopene/vitamin E supplementation, compared with lycopene alone. These observations were probably due to the synergistic effect of lycopene and vitamin E against oxidative stress injury, as suggested by Siler et al. [40] and Limpens et al. [41]. To explain oxidative stress as a possible contributor to tau pathology, tau phosphorylation was examined in P301L mice with lycopene alone or lycopene/vitamin E supplementations. Indeed, lycopene alone could reduce tau phosphorylation at Ser262 site and lycopene/vitamin E combination decreased the phosphorylation of tau protein at Ser262 and Ser396 epitopes, while lycopene and vitamin E combinations had a lower extent at Ser396 epitope compared with lycopene alone. The phosphorylation of tau plays a physiological role in regulating the affinity of tau with microtubules. Hyperphosphorylated tau is prone to detach from microtubules and to aggregate NFTs, numbers of which in the neocortex positively correlate with the severity of cognitive decline [42]. However, hyperphosphorylation of tau protein in the brain of P301L mice can be alleviated by lycopene alone or lycopene/vitamin E combination in our study. Observed improvements, including passive and spatial memory enhancements, were predominantly found in P301L mice supplemented with lycopene or lycopene/vitamin E. Moreover, lycopene in combination with vitamin E seemed more effective on improvements in behavioral performances. These data suggest that the neuroprotective effects of lycopene alone and lycopene/vitamin E in combination could prevent cognitive decline of P301L mice by anti-hyperphosphorylation of tau protein via inhibiting oxidative stress.

To summarize, our study demonstrates for the first time that lycopene regulates the phosphorylation of tau protein by the oxidative stress pathway, consequently affecting cognitive function of transgenic mouse models of tauopathy. Although tau-associated neurodegenerative diseases are probably associated with multiple etiologies and pathophysiologic mechanisms, oxidative stress appears to be interlinked with tau pathology throughout the pathophysiologic process of tauopathies. Accordingly, antioxidant treatments work well in the retardation of the progression of tauopathies.

Footnotes

ACKNOWLEDGMENTS

Thanks to Prof. Xuelan Wang for providing the P301L transgenic mice.

This work was financially supported by a grant from Nutrition and Science foundation of BY-HEALTH Co., Ltd (No. TY0141101) and the Tianjin Research Program of Application Foundation and Advanced Technology (No. 13JCZDJC30400).

Authors’ disclosures available online ().

References

Spillantini

, Goedert

(2013) Tau pathology and neurodegeneration. Lancet Neurol 12, 609–622.

Chan

, Wang

, Wu

, Liu

, Theodoratou

, Car

, Middleton

, Russ

, Deary

, Campbell

, Wang

, Rudan

(2013) Epidemiology of Alzheimer’s disease and other forms of dementia in China, 1990–2010: A systematic review and analysis. Lancet 381, 2016–2023.

Wolfe

(2012) The role of tau in neurodegenerative diseases and its potential as a therapeutic target. Scientifica (Cairo) 2012, 796024.

Alavi Naini

, Soussi-Yanicostas

(2015) Tau hyperphosphorylation and oxidative stress, a critical vicious circle in neurodegenerative tauopathies? Oxid Med Cell Longev 2015, 151979.

Moosmann

, Behl

(2002) Antioxidants as treatment for neurodegenerative disorders. Expert Opin Investig Drugs 11, 1407–1435.

Martínez

, Carmona

, Portero-Otin

, Naudí

, Pamplona

, Ferrer

(2008) Type-dependent oxidative damage in frontotemporal lobar degeneration: Cortical astrocytes are targets of oxidative damage. J Neuropathol Exp Neurol 67, 1122–1136.

Litvan

(2004) Update on progressive supranuclear palsy. Curr Neurol Neurosci Rep 4, 296.

Liu

C-C

, Huang

C-C

, Lin

W-T

, Hsieh

C-C

, Huang

S-Y

, Lin

S-J

, Yang

S-C

(2005) Lycopene supplementation attenuated xanthine oxidase and myeloperoxidase activities in skeletal muscle tissues of rats after exhaustive exercise. Br J Nutr 94, 595–601.

Min

, Min

(2014) Serum lycopene, lutein and zeaxanthin, and the risk of Alzheimer’s disease mortality in older adults. Dement Geriatr Cogn Disord 37, 246–256.

10.

Dias

IHK

, Polidori

, Li

, Weber

, Stahl

, Nelles

, Grune

, Griffiths

(2014) Plasma levels of HDL and carotenoids are lower in dementia patients with vascular comorbidities. J Alzheimers Dis 40, 399–408.

11.

Giavarotti

, Simon

, Azzalis

, Fonseca

FLA

, Lima

, Freitas

MCV

, Brunialti

MKC

, Salomão

, Moscardi

AAVS

, Montaño

MBMM

, Ramos

, Junqueira

VBC

(2013) Mild systemic oxidative stress in the subclinical stage of Alzheimer’s disease. Oxid Med Cell Longev 2013, 609019.

12.

Vance

, Su

, Fontham

ETH

, Koo

, Chun

(2013) Dietary antioxidants and prostate cancer: A review. Nutr Cancer 65, 793–801.

13.

Fuhrman

, Volkova

, Rosenblat

, Aviram

(2000) Lycopene synergistically inhibits LDL oxidation in combination with vitamin E, glabridin, rosmarinic acid, carnosic acid, or garlic. Antioxid Redox Signal 2, 491–506.

14.

Schroeder

, Becker

, Skibsted

(2006) Molecular mechanism of antioxidant synergism of tocotrienols and carotenoids in palm oil. J Agric Food Chem 54, 3445–3453.

15.

Crichton

, Bryan

, Murphy

(2013) Dietary antioxidants, cognitive function and dementia–a systematic review. Plant Foods Hum Nutr 68, 279–292.

16.

Obulesu

, Dowlathabad

, Bramhachari

(2011) Carotenoids and Alzheimer’s disease: An insight into therapeutic role of retinoids in animal models. Neurochem Int 59, 535–541.

17.

, Ding

, Zhu

, Chen

, Wang

(2016) Olfactory dysfunctions and decreased nitric oxide production in the brain of human P301L tau transgenic mice. Neurochem Res 41, 722–730.

18.

Götz

, Chen

, Barmettler

, Nitsch

(2001) Tau filament formation in transgenic mice expressing P301L tau. J Biol Chem 276, 529–534.

19.

, Chen

, Wang

, Xiao

, Hong

(2016) Multi-vitamin B supplementation reverses hypoxia-induced tau hyperphosphorylation and improves memory function in adult mice. J Alzheimers Dis 54, 297–306.

20.

Götz

, Ittner

(2008) Animal models of Alzheimer’s disease and frontotemporal dementia. Nat Rev Neurosci 9, 532–544.

21.

Deters

, Ittner

, Götz

(2008) Divergent phosphorylation pattern of tau in P301L tau transgenic mice. Eur J Neurosci 28, 137–147.

22.

Schulz

, Eckert

, Rhein

, Mai

, Haase

, Reichert

, Jendrach

, Müller

, Leuner

(2012) A new link to mitochondrial impairment in tauopathies. Mol Neurobiol 46, 205–216.

23.

Cente

, Filipcik

, Pevalova

, Novak

(2006) Expression of a truncated tau protein induces oxidative stress in a rodent model of tauopathy. Eur J Neurosci 24, 1085–1090.

24.

Torres

, Quaglio

, de Souza

, Garcia

, Dati

LMM

, Moreira

, Loureiro

, de

, de Souza-Talarico

, Smid

, Porto

, Bottino

, de

, Nitrini

, Barros

, de

, Camarini

, Marcourakis

(2011) Peripheral oxidative stress biomarkers in mild cognitive impairment and Alzheimer’s disease. J Alzheimers Dis 26, 59–68.

25.

Skoumalová

, Hort

(2012) Blood markers of oxidative stress in Alzheimer’s disease. J Cell Mol Med 16, 2291–2300.

26.

Keller

, Schmitt

, Scheff

, Ding

, Chen

, Butterfield

, Markesbery

(2005) Evidence of increased oxidative damage in subjects with mild cognitive impairment. Neurology 64, 1152–1156.

27.

Ansari

, Scheff

(2010) Oxidative stress in the progression of Alzheimer disease in the frontal cortex. J Neuropathol Exp Neurol 69, 155–167.

28.

Padurariu

, Ciobica

, Hritcu

, Stoica

, Bild

, Stefanescu

(2010) Changes of some oxidative stress markers in the serum of patients with mild cognitive impairment and Alzheimer’s disease. Neurosci Lett 469, 6–10.

29.

Melov

, Adlard

, Morten

, Johnson

, Golden

, Hinerfeld

, Schilling

, Mavros

, Masters

, Volitakis

, Li

Q-X

, Laughton

, Hubbard

, Cherny

, Gibson

, Bush

(2007) Mitochondrial oxidative stress causes hyperphosphorylation of tau. PloS One 2, e536.

30.

Arthur

(2000) The glutathione peroxidases. Cell Mol Life Sci 57, 1825–1835.

31.

Dias-Santagata

, Fulga

, Duttaroy

, Feany

(2007) Oxidative stress mediates tau-induced neurodegeneration in Drosophila. J Clin Invest 117, 236–245.

32.

, Wang

, Lee

H-G

, Tabaton

, Perry

, Smith

, Zhu

(2010) Chronic oxidative stress causes increased tau phosphorylation in M17 neuroblastoma cells. Neurosci Lett 468, 267–271.

33.

Pérez

, Cuadros

, Smith

, Perry

, Avila

(2000) Phosphorylated, but not native, tau protein assembles following reaction with the lipid peroxidation product, 4-hydroxy-2-nonenal. FEBS Lett 486, 270–274.

34.

Gómez-Ramos

, Díaz-Nido

, Smith

, Perry

, Avila

(2003) Effect of the lipid peroxidation product acrolein on tau phosphorylation in neural cells. J Neurosci Res 71, 863–870.

35.

Dumont

, Stack

, Elipenahli

, Jainuddin

, Gerges

, Starkova

, Yang

, Starkov

, Beal

(2011) Behavioral deficit, oxidative stress, and mitochondrial dysfunction precede tau pathology in P301S transgenic mice. FASEB J 25, 4063–4072.

36.

Starkov

(2008) The role of mitochondria in reactive oxygen species metabolism and signaling. Ann N Y Acad Sci 1147, 37–52.

37.

Wang

X-D

(2012) Lycopene metabolism and its biological significance. Am J Clin Nutr 96, 1214S–22S.

38.

Friedman

(2013) Anticarcinogenic, cardioprotective, and other health benefits of tomato compounds lycopene, α-tomatine, and tomatidine in pure form and in fresh and processed tomatoes. J Agric Food Chem 61, 9534–9550.

39.

Del Giudice

, Petruk

, Raiola

, Barone

, Monti

, Rigano

(2016) Carotenoids in fresh and processed tomato (Solanum lycopersicum) fruits protect cells from oxidative stress injury. J Sci Food Agric, doi: 10.1002/jsfa.7910 [epub ahead of print].

40.

Siler

, Barella

, Spitzer

, Schnorr

, Lein

, Goralczyk

, Wertz

(2004) Lycopene and vitamin E interfere with autocrine/paracrine loops in the Dunning prostate cancer model. FASEB J 18, 1019–1021.

41.

Limpens

, Schröder

, de Ridder

CMA

, Bolder

, Wildhagen

, Obermüller-Jevic

, Krämer

, van Weerden

(2006) Combined lycopene and vitamin E treatment suppresses the growth of PC-346C human prostate cancer cells in nude mice. J Nutr 136, 1287–1293.

42.

Arriagada

, Growdon

, Hedley-Whyte

, Hyman

(1992) Neurofibrillary tangles but not senile plaques parallel duration and severity of Alzheimer’s disease. Neurology 42, 631–639.